Compositions comprising digestive enzymes

ABSTRACT

Provided herein are compositions with enhanced protein specific activity, protein combinations and methods for the preparation thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of International PatentApplication No. PCT/US2020/045519, filed Aug. 7, 2020, which claims thebenefit of U.S. Provisional Application No. 62/883,800, filed Aug. 7,2019 and U.S. Provisional Application No. 62/941,627, filed Nov. 27,2019; each of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 3, 2020, isnamed 49160-716.601_ST25.txt and is 33,680 bytes in size.

BACKGROUND OF THE INVENTION

Pepsin is a protease that cleaves polypeptides into smaller units. Innature, pepsin is a digestive enzyme found in the stomach of animals andhumans, that helps to digest food. Outside of its naturally-occurringenvironment, pepsin is used as a processing enzyme in a variety ofapplications. For example, pepsin can be used to modify foodingredients, it is a component in cheese making, used in the leatherindustry and also used to prepare antibody fragments used forpharmaceutical and biotechnology applications.

Pepsin is expressed as a zymogen pepsinogen, which has additional aminoacids as compared with pepsin. Under non-acidic pH condition, pepsinogenis activimmature, due to the presence of the propeptide. Under acidic pHconditions, pepsinogen can unfold and cleave itself to create the matureform of the enzyme which is pepsin. Typically, the enzyme is extractedfrom pig stomach. Because the stomach is an acidic environment, theextracted form is primarily the cleaved and active pepsin form.

SUMMARY OF THE INVENTION

An aspect of the present disclosure is a method of producing ahigh-activity stable pepsin composition. The method comprises steps of(a) providing a microorganism that expresses a recombinant pepsinogen;(b) culturing the microorganism under conditions in which therecombinant pepsinogen polypeptide is expressed and secreted by themicroorganism into a growth media; (c) harvesting the growth media andremoving the microorganism therefrom to obtain a liquid startingmaterial; (d) lowering the pH of the liquid starting material to lessthan pH 4.0 to obtain an activated pepsin composition; and (e) raisingthe pH of the activated pepsin composition to a pH of between about 5.4and 7.0 to obtain a high-activity stable pepsin composition.

In some embodiments, the method further comprises, after step (d), astep of isolating activated pepsin polypeptide from other proteins andsmall molecules in the activated pepsin composition and/or, after step(e), a step of isolating activated pepsin polypeptide from otherproteins and small molecules in the high-activity stable pepsincomposition.

In some embodiments, the high-activity stable pepsin compositioncomprises an intact and stable proteolytically inactive form ofrecombinant pepsin.

In some embodiments, the high-activity stable pepsin composition has aspecific activity at pH of 2 of at least 20,000 FCC units/mg totalprotein, e.g., greater than 30,000 FCC units/mg total protein, greaterthan 40,000 FCC units/mg total protein, greater than 50,000 FCC units/mgtotal protein, greater than 60,000 FCC units/mg total protein, orgreater than 65,000 FCC units/mg total protein. In embodiments, an FCCunit (also referred to herein as a pepsin unit) is defined as thatquantity of enzyme that produces the equivalent of 1 μmol of tyrosineper min under the conditions of incubating the enzyme with 2% hemoglobinsubstrate at pH 1.6 for 10 minutes at 25° C. (see Food Chemical Codex,11th ed. (Pharmacopeial Convention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, the final pH of the high-activity stable pepsincomposition is between about 5.4 and 6.0.

In some embodiments, the recombinant pepsinogen is present in at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or greater than 20 g/liter in the growth medium prior to step (c).

In some embodiments, the growth medium is filtered after step (b), theliquid starting material is filtered after step (c), the activatedpepsin composition is filtered after step (d), and/or the high-activitystable pepsin composition is filtered after step (e).

In some embodiments, a recombinant pepsin in the high-activity stablepepsin composition comprises an amino acid sequence of a pig, a sheep, aCentral European red deer, a goat, a cow, a human, a yak, or a zebupepsin.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of a pig, a sheep, a Central European red deer, a goat, a cow,a human, a yak, or a zebu pepsinogen.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of one of SEQ ID Nos. 1-9 or a sequence with at least 90%identity thereto.

In some embodiments, the microorganism is selected from yeast,filamentous fungi, a saccharomyces species, a bacteria, a Pichiaspecies, a Trichoderma species and an Aspergillus species.

In some embodiments, the microorganism that expresses the recombinantpepsinogen comprises a first inducible promoter which regulates theexpression of the recombinant pepsinogen. In some cases, the methodfurther comprises a step of inducing the expression of the recombinantpepsinogen after or at least partially concurrent with the step ofculturing the microorganism. In some cases, the microorganism is aPichia species. In some embodiments, the microorganism further comprisesa helper factor, e.g., a helper factor that is expressed from a secondinducible promoter. In some cases, the first inducible promoter, thesecond inducible promoter, or both the first and second induciblepromoters are induced by methanol.

In some embodiments, the method further comprises a desalting step afterthe harvesting step and/or the method further comprises a drying stepafter the harvesting step. The drying step may be spray drying orlyophilization.

In some embodiments, the high-activity stable pepsin compositioncomprises a recombinant pepsin having an amino acid sequence of SEQ IDNO. 10 or a sequence with at least 90% identity thereto.

Another aspect of the present disclosure is a method of producing arecombinant pepsinogen. The method including steps of (a) providing amicroorganism that expresses a recombinant pepsinogen, in which theexpressed pepsinogen is secreted by the microorganism into a growthmedia; (b) culturing the microorganism until the secreted pepsinogen ispresent in the growth medium in an amount of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greaterthan 20 g/liter; and (c) harvesting the growth media and removing themicroorganism therefrom to obtain a liquid material in which therecombinant pepsinogen is substantially in a proteolytically inactiveform.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of a pig, a sheep, a Central European red deer, a goat, a cow,a human, a yak, or a zebu pepsinogen.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of one of SEQ ID Nos. 1-9 or a sequence with at least 90%identity thereto.

In some embodiments, the microorganism is selected from yeast,filamentous fungi, a saccharomyces species, a bacteria, a Pichiaspecies, a Trichoderma species and an Aspergillus species.

In some embodiments, the microorganism that expresses the recombinantpepsinogen comprises a first inducible promoter which regulates theexpression of the recombinant pepsinogen. In some cases, the methodfurther comprises a step of inducing the expression of the recombinantpepsinogen after or at least partially concurrent with the step ofculturing the microorganism. In some cases, the microorganism is aPichia species. In some embodiments, the microorganism further comprisesa helper factor, e.g., a helper factor that is expressed from a secondinducible promoter. In some cases, the first inducible promoter, thesecond inducible promoter, or both the first and second induciblepromoters are induced by methanol.

In some embodiments, the method further comprises a desalting step afterthe harvesting step and/or the method further comprises a drying stepafter the harvesting step. The drying step may be spray drying orlyophilization.

In some embodiments, the recombinant pepsinogen that is substantially inthe proteolytically inactive form is capable of being activated byreducing the pH or by exposure to an acidic environment to produce ahigh-activity stable pepsin composition, in which the high-activitystable pepsin composition has a specific activity at pH of 2 of at least20,000 FCC units/mg total protein, e.g., greater than 30,000 FCCunits/mg total protein, greater than 40,000 FCC units/mg total protein,greater than 50,000 FCC units/mg total protein, greater than 60,000 FCCunits/mg total protein, or greater than 65,000 FCC units/mg totalprotein. In embodiments, an FCC unit (also referred to herein as apepsin unit) is defined as that quantity of enzyme that produces theequivalent of 1 μmol of tyrosine per min under the conditions ofincubating the enzyme with 2% hemoglobin substrate at pH 1.6 for 10minutes at 25° C. (see Food Chemical Codex, 11th ed. (PharmacopeialConvention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, the high-activity stable pepsin compositioncomprises a recombinant pepsin having an amino acid sequence of SEQ IDNO. 10 or a sequence with at least 90% identity thereto.

In some embodiments, the method further comprises formulating therecombinant pepsinogen with at least one ingredient to create aformulated recombinant pepsinogen composition in liquid (e.g., syrup andgel), powder, pill, tablet or capsule form.

Yet another aspect of the present disclosure is a method of treating adisease or condition of the gastrointestinal tract. The method includingsteps of (a) providing the recombinant pepsinogen in a formulatedcomposition, in which the pepsinogen is produced by the method accordingto any herein disclosed method and (b) administering the formulatedcomposition for oral administration; in which upon contact of theformulated composition with an animal gut environment, the pepsinogen isconverted to high-activity stable pepsin; and in which the pepsin iseffective to treat the disease or condition of the gastrointestinaltract.

In an aspect, the present disclosure provides a composition comprising arecombinant pepsin polypeptide. The composition is (a) free fromanimal-derived proteins, (b) the pepsin polypeptide is substantially inan intact and stable proteolytically inactive form, (c) the compositionhas a pH greater than about 5.4, and (d) the composition has a specificactivity of at least 20,000 FCC units/mg total protein.

In some embodiments, the composition has a specific activity of at least30,000 FCC units/mg total protein, at least 40,000 FCC units/mg totalprotein, at least 50,000 FCC units/mg total protein, at least 60,000 FCCunits/mg total protein, or at least 70,000 FCC units/mg total protein.

In some embodiments, the composition has a pH between about 5.4 to about6.0.

In some embodiments, the proteolytically inactive pepsin polypeptideform is stable in the composition for at least 6 months at roomtemperature. In some embodiments, the proteolytically inactive pepsinpolypeptide form is stable in the composition for at least 6 months at4° C. In some embodiments, the composition comprises a protein contentof at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or 90% w/w.

In some embodiments, the recombinant pepsin polypeptide comprises anamino acid sequence of a pig, a sheep, a Central European red deer, agoat, a cow, a human, a yak, or a zebu pepsin.

In some embodiments, the recombinant pepsin polypeptide comprises SEQ IDNO: 10, or an amino acid sequence with at least 90% identity thereto.

In some embodiments, the recombinant pepsin polypeptide is produced in ayeast, a filamentous fungi, a saccharomyces species, a bacteria, aPichia species, a Trichoderma species, or an Aspergillus species. Insome cases, the recombinant pepsin polypeptide is produced in a Pichiasp.

In some embodiments, the composition is in powdered form. In someembodiments, the proteolytically inactive pepsin polypeptide form isstable in the powdered composition for at least 6 months at roomtemperature. In some embodiments, the proteolytically inactive pepsinpolypeptide form is stable in the powdered composition for at least 6months at 4° C. In some embodiments, the powdered composition comprisesa protein content of at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w. In some embodiments, thepowdered composition has a moisture content of less than about 10%. Insome embodiments, the recombinant pepsin polypeptide is produced in ayeast, a filamentous fungi, a saccharomyces species, a bacteria, aPichia species, a Trichoderma species, or an Aspergillus species. Insome cases, the recombinant pepsin polypeptide is produced in a Pichiasp.

In some embodiments, the composition is in liquid form. In a liquidcomposition, the concentration of the recombinant pepsin polypeptide maybe at least 2 g per liter, at least 5 g per liter, at least 7 g perliter, at least 10 g per liter, at least 15 g per liter, or at least 20g per liter. In some embodiments, the proteolytically inactive pepsinpolypeptide form is stable in the liquid composition for at least 30days at a temperature of about 4° C. In some embodiments, therecombinant pepsin polypeptide is produced in a yeast, a filamentousfungi, a saccharomyces species, a bacteria, a Pichia species, aTichoderma species, or an Aspergillus species. In some cases, therecombinant pepsin polypeptide is produced in a Pichia sp.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsin.

In an aspect, the present disclosure provides a composition comprising arecombinant pepsinogen polypeptide. The composition is (a) free fromanimal-derived proteins, (b) the pepsinogen polypeptide is substantiallyin an intact and stable proteolytically inactive form, (c) thecomposition has a pH greater than about 5.4, and (d) the pepsinogenpolypeptide is capable of being activated to a proteolytically activepepsin form that has a specific activity of at least 20,000 FCC units/mgtotal protein.

In some embodiments, the specific activity is at least 30,000 FCCunits/mg total protein, at least 40,000 FCC units/mg total protein, atleast 50,000 FCC units/mg total protein, at least 60,000 FCC units/mgtotal protein, or at least 70,000 FCC units/mg total protein.

In some embodiments, the composition is a liquid and the recombinantpepsinogen polypeptide is present in the composition at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 orgreater than 20 g/liter.

In some embodiments, the composition is a powder.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of one of SEQ ID NOs: 1-9, or an amino acid sequence with atleast 90% identity thereto. In some cases, the amount of pepsin in thecomposition is less than 10%, 5%, 1%, 0.5%, 0.1% or 0.05% (weightpepsin/weight pepsinogen).

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsinogen.

In some embodiments, the composition comprises a protein content of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% w/w.

In some embodiments, the composition further comprises at least oneingredient to create a formulated recombinant pepsinogen composition inliquid (e.g., syrup and gel), powder, pill, tablet or capsule form. Theformulated composition may be substantially devoid of pepsin. Thepepsinogen is capable of activation when exposed to an animal gutenvironment and/or to a pH of about 2.

In some embodiments, the recombinant pepsinogen polypeptide is producedin a yeast, a filamentous fungi, a saccharomyces species, a bacteria, aPichia species, a Trichoderma species or an Aspergillus species. In somecases, the recombinant pepsinogen polypeptide is produced in a Pichiasp.

An aspect of the present disclosure is a method of producing a stablepepsin composition. The method comprises steps of (a) providing amicroorganism that expresses a recombinant pepsinogen; (b) culturing themicroorganism under conditions in which the recombinant pepsinogenpolypeptide is expressed and secreted by the microorganism into a growthmedia; (c) harvesting the growth media and removing the microorganismtherefrom to obtain a liquid starting material; (d) lowering the pH ofthe liquid starting material to less than pH 4.0 to obtain an activatedpepsin composition; and (e) raising the pH of the activated pepsincomposition to a pH of between about 5.4 and 7.0 to obtain a stablepepsin composition. The stable pepsin composition has a specificactivity at pH of 2 of between about 100 FCC units/mg total protein andabout 19,000 FCC units/mg total protein.

In some embodiments, the method further comprises, after step (d), astep of isolating activated pepsin polypeptide from other proteins andsmall molecules in the activated pepsin composition and/or, after step(e), a step of isolating activated pepsin polypeptide from otherproteins and small molecules in the stable pepsin composition.

In some embodiments, the stable pepsin composition comprises an intactand stable proteolytically inactive form of recombinant pepsin.

In some embodiments, the specific activity at pH of 2 is at least 500FCC units/mg total protein, at least 1000 FCC units/mg total protein, atleast 2000 FCC units/mg total protein, at least 5000 FCC units/mg totalprotein, at least 10000 FCC units/mg total protein, or at least 15000FCC units/mg total protein, and less than 20000 FCC units/mg totalprotein. As examples, the specific activity at pH of 2 is at least 100FCC units/mg total protein, at least 200 FCC units/mg total protein, atleast 300 FCC units/mg total protein, at least 400 FCC units/mg totalprotein, at least 500 FCC units/mg total protein, at least 600 FCCunits/mg total protein, at least 700 FCC units/mg total protein, atleast 800 FCC units/mg total protein, at least 900 FCC units/mg totalprotein, at least 1000 FCC units/mg total protein, at least 1100 FCCunits/mg total protein, at least 1200 FCC units/mg total protein, atleast 1300 FCC units/mg total protein, at least 1400 FCC units/mg totalprotein, at least 1500 FCC units/mg total protein, at least 1600 FCCunits/mg total protein, at least 1700 FCC units/mg total protein, atleast 1800 FCC units/mg total protein, at least 1900 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 3000 FCCunits/mg total protein, at least 4000 FCC units/mg total protein, atleast 5000 FCC units/mg total protein, at least 6000 FCC units/mg totalprotein, at least 7000 FCC units/mg total protein, at least 8000 FCCunits/mg total protein, at least 9000 FCC units/mg total protein, atleast 10000 FCC units/mg total protein, at least 11000 FCC units/mgtotal protein, at least 12000 FCC units/mg total protein, at least 13000FCC units/mg total protein, at least 14000 FCC units/mg total protein,at least 15000 FCC units/mg total protein, at least 16000 FCC units/mgtotal protein, at least 17000 FCC units/mg total protein, at least 18000FCC units/mg total protein, or at least 19000 FCC units/mg total proteinand less than 20000 FCC units/mg total protein.

In some embodiments, the final pH of the stable pepsin composition isbetween about 5.4 and 6.0.

In some embodiments, the recombinant pepsinogen is present in at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or greater than 20 g/liter in the growth medium prior to step (c).

In some embodiments, the growth medium is filtered after step (b), theliquid starting material is filtered after step (c), the activatedpepsin composition is filtered after step (d), and/or the stable pepsincomposition is filtered after step (e).

In some embodiments, a recombinant pepsin in the stable pepsincomposition comprises an amino acid sequence of a pig, a sheep, aCentral European red deer, a goat, a cow, a human, a yak, or a zebupepsin.

Another aspect of the present disclosure is a method of producing arecombinant pepsinogen. The method comprises steps of (a) providing amicroorganism that expresses a recombinant pepsinogen, wherein theexpressed pepsinogen is secreted by the microorganism into a growthmedia; (b) culturing the microorganism until the secreted pepsinogen ispresent in the growth medium in an amount of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greaterthan 20 g/liter; and (c) harvesting the growth media and removing themicroorganism therefrom to obtain a liquid material in which therecombinant pepsinogen is substantially in a proteolytically inactiveform. The recombinant pepsinogen that is substantially in theproteolytically inactive form is capable of being activated by reducingthe pH or by exposure to an acidic environment to produce a stablepepsin composition, wherein the stable pepsin composition has a specificactivity at pH of 2 of between about 100 FCC units/mg total protein andabout 19,000 FCC units/mg total protein.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of a pig, a sheep, a Central European red deer, a goat, a cow,a human, a yak, or a zebu pepsinogen.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of one of SEQ ID Nos. 1-9 or a sequence with at least 90%identity thereto.

In some embodiments, the microorganism that expresses the recombinantpepsinogen comprises a first inducible promoter which regulates theexpression of the recombinant pepsinogen. In some embodiments, the themethod further comprises a step of inducing the expression of therecombinant pepsinogen after or at least partially concurrent with thestep of culturing the microorganism.

In some embodiments, the microorganism is selected from yeast,filamentous fungi, a saccharomyces species, a bacteria, a Pichiaspecies, a Trichoderma species and an Aspergillus species. In someembodiments, the microorganism is a Pichia species. In some embodiments,the microorganism further comprises a helper factor. In someembodiments, the helper factor is expressed from a second induciblepromoter. In some embodiments, the first inducible promoter, the secondinducible promoter, or both the first and second inducible promoters areinduced by methanol.

In some embodiments, the method further comprises a desalting step afterthe harvesting step and/or a drying step after the harvesting step. Insome embodiments, the drying step comprises spray drying orlyophilization.

In some embodiments, the specific activity at pH of 2 is at least 500FCC units/mg total protein, at least 1000 FCC units/mg total protein, atleast 2000 FCC units/mg total protein, at least 5000 FCC units/mg totalprotein, at least 10000 FCC units/mg total protein, or at least 15000FCC units/mg total protein, and less than 20000 FCC units/mg totalprotein. As examples, the specific activity at pH of 2 is at least 100FCC units/mg total protein, at least 200 FCC units/mg total protein, atleast 300 FCC units/mg total protein, at least 400 FCC units/mg totalprotein, at least 500 FCC units/mg total protein, at least 600 FCCunits/mg total protein, at least 700 FCC units/mg total protein, atleast 800 FCC units/mg total protein, at least 900 FCC units/mg totalprotein, at least 1000 FCC units/mg total protein, at least 1100 FCCunits/mg total protein, at least 1200 FCC units/mg total protein, atleast 1300 FCC units/mg total protein, at least 1400 FCC units/mg totalprotein, at least 1500 FCC units/mg total protein, at least 1600 FCCunits/mg total protein, at least 1700 FCC units/mg total protein, atleast 1800 FCC units/mg total protein, at least 1900 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 3000 FCCunits/mg total protein, at least 4000 FCC units/mg total protein, atleast 5000 FCC units/mg total protein, at least 6000 FCC units/mg totalprotein, at least 7000 FCC units/mg total protein, at least 8000 FCCunits/mg total protein, at least 9000 FCC units/mg total protein, atleast 10000 FCC units/mg total protein, at least 11000 FCC units/mgtotal protein, at least 12000 FCC units/mg total protein, at least 13000FCC units/mg total protein, at least 14000 FCC units/mg total protein,at least 15000 FCC units/mg total protein, at least 16000 FCC units/mgtotal protein, at least 17000 FCC units/mg total protein, at least 18000FCC units/mg total protein, or at least 19000 FCC units/mg total proteinand less than 20000 FCC units/mg total protein.

In some embodiments, the stable pepsin composition comprises arecombinant pepsin having an amino acid sequence of SEQ ID NO. 10 or asequence with at least 90% identity thereto.

In some embodiments, the method further comprising formulating therecombinant pepsin or recombinant pepsinogen with at least oneingredient to create, respectively, a formulated recombinant pepsincomposition or recombinant pepsinogen composition in liquid, powder,pill, tablet, or capsule form. In some embodiments, the liquid is asyrup or a gel

Yet another aspect of the present disclosure is composition comprising arecombinant pepsin polypeptide. The composition is (a) free fromanimal-derived proteins, (b) the pepsin polypeptide is substantially inan intact and stable proteolytically inactive form, (c) the compositionhas a pH greater than about 5.4, and (d) the composition has a specificactivity of between about 100 FCC units/mg total protein and about19,000 FCC units/mg total protein.

In some embodiments, the specific activity at pH of 2 is at least 500FCC units/mg total protein, at least 1000 FCC units/mg total protein, atleast 2000 FCC units/mg total protein, at least 5000 FCC units/mg totalprotein, at least 10000 FCC units/mg total protein, or at least 15000FCC units/mg total protein, and less than 20000 FCC units/mg totalprotein. In some embodiments, the specific activity at pH of 2 is atleast 500 FCC units/mg total protein, at least 1000 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 5000 FCCunits/mg total protein, at least 10000 FCC units/mg total protein, or atleast 15000 FCC units/mg total protein, and less than 20000 FCC units/mgtotal protein. As examples, the specific activity at pH of 2 is at least100 FCC units/mg total protein, at least 200 FCC units/mg total protein,at least 300 FCC units/mg total protein, at least 400 FCC units/mg totalprotein, at least 500 FCC units/mg total protein, at least 600 FCCunits/mg total protein, at least 700 FCC units/mg total protein, atleast 800 FCC units/mg total protein, at least 900 FCC units/mg totalprotein, at least 1000 FCC units/mg total protein, at least 1100 FCCunits/mg total protein, at least 1200 FCC units/mg total protein, atleast 1300 FCC units/mg total protein, at least 1400 FCC units/mg totalprotein, at least 1500 FCC units/mg total protein, at least 1600 FCCunits/mg total protein, at least 1700 FCC units/mg total protein, atleast 1800 FCC units/mg total protein, at least 1900 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 3000 FCCunits/mg total protein, at least 4000 FCC units/mg total protein, atleast 5000 FCC units/mg total protein, at least 6000 FCC units/mg totalprotein, at least 7000 FCC units/mg total protein, at least 8000 FCCunits/mg total protein, at least 9000 FCC units/mg total protein, atleast 10000 FCC units/mg total protein, at least 11000 FCC units/mgtotal protein, at least 12000 FCC units/mg total protein, at least 13000FCC units/mg total protein, at least 14000 FCC units/mg total protein,at least 15000 FCC units/mg total protein, at least 16000 FCC units/mgtotal protein, at least 17000 FCC units/mg total protein, at least 18000FCC units/mg total protein, or at least 19000 FCC units/mg total proteinand less than 20000 FCC units/mg total protein.

In some embodiments, the composition has a pH between about 5.4 to about6.0.

In some embodiments, the composition comprises a protein content of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% w/w.

In some embodiments, the composition is in powdered form.

In some embodiments, the composition is in liquid form.

In some embodiments, the recombinant pepsin polypeptide is at least 10mg per liter, at least 100 mg per liter, at least 500 mg per liter, atleast 1 g per liter, at least 2 g per liter, at least 5 g per liter, atleast 7 g per liter, at least 10 g per liter, at least 15 g per liter,or at least 20 g per liter. As examples, the recombinant pepsinpolypeptide is at least 10 mg per liter, at least 20 mg per liter, atleast 30 mg per liter, at least 40 mg per liter, at least 50 mg perliter, at least 60 mg per liter, at least 70 mg per liter, at least 80mg per liter, at least 90 mg per liter, at least 100 mg per liter, atleast 200 mg per liter, at least 300 mg per liter, at least 400 mg perliter, at least 500 mg per liter, at least 600 mg per liter, at least700 mg per liter, at least 800 mg per liter, at least 900 mg per liter,at least 1000 mg per liter, at least 1100 mg per liter, at least 1200 mgper liter, at least 1300 mg per liter, at least 1400 mg per liter, atleast 1500 mg per liter, at least 1600 mg per liter, at least 1700 mgper liter, at least 1800 mg per liter, or at least 1900 mg per liter.

In some embodiments, the recombinant pepsin polypeptide comprises anamino acid sequence of a pig, a sheep, a Central European red deer, agoat, a cow, a human, a yak, or a zebu pepsin.

In some embodiments, the recombinant pepsin polypeptide comprises SEQ IDNO: 10, or an amino acid sequence with at least 90% identity thereto.

In some embodiments, the recombinant pepsin polypeptide is produced in ayeast, a filamentous fungi, a saccharomyces species, a bacteria, aPichia species, a Trichoderma species, or an Aspergillus species. Insome embodiments, the recombinant pepsin polypeptide is produced in aPichia sp.

In some embodiments, the liquid form is a syrup or a gel.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsin.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

Additionally, any composition or method disclosed herein is applicableto any herein-disclosed composition or method. In other words, anyaspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows pepsinogen composition at pH 5.95 (lane B) compared toactivated pepsin at pH 3.5 at various temperatures after incubation withHCl for 1 minute, 15 minutes, 30 minutes, 45 minutes and 60 minutes.

FIG. 2 shows pepsinogen composition at pH 5.95 (lane B) compared toactivated pepsin at various pH after incubation with HCl for 5 minutes,15 minutes, 30 minutes, and 120 minutes.

FIG. 3 shows pepsin activation for various times before conversion toinactive pepsin at pH 6.33 at 37° C.

FIG. 4 shows pepsinogen composition activated at temperatures of 45° C.and 55° C. for various times.

FIG. 5A shows a graph comparing the pH profile of a recombinant pepsincomposition and a commercially-available native porcine pepsin. In thisexperiment, activity is expressed on the graph as FCC units/unit pepsin,where each pepsin unit is defined as the amount of pepsin present in thesample, derived from its peak area determined by HPLC. FIG. 5B shows agraph comparing the activity over a pH range of the recombinant pepsincomposition and the commercially-available native porcine pepsinpresented as the activity per mg powder composition.

FIG. 6 shows a western blot comparing sizes of a recombinant pepsincomposition and a commercially-available native porcine pepsin.

FIG. 7A to FIG. 7C show gels showing the absence of nucleic acidencoding pepsinogen in recombinant pepsin compositions, as compared topositive controls of plasmid DNA containing a nucleic acid encodingpepsinogen.

FIG. 8 shows a graph showing the lack of animal derived proteins in therecombinant pepsin composition as compared to a commercially-availablenative porcine pepsin.

FIG. 9 shows a graph characterizing stability of a liquid recombinantpepsin composition over time.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions and methods for making compositionscomprising pepsin or comprising pepsinogen.

Expression of Pepsinogen

The protein pepsinogen refers to an immature form of the protein pepsinand carries a propeptide. Upon maturation, the propeptide is cleaved offto produce pepsin. The mature form pepsin may be enzymatically activeunder certain conditions, such as low pH. In some embodiments,compositions containing the mature form of a recombinant pepsin, whenplaced under activation conditions, provide a high level of enzymaticactivity.

Provided herein are methods for producing compositions of recombinantpepsin that are stable and can be activated to a high level of specificactivity.

An aspect of the present disclosure is a method of producing ahigh-activity stable pepsin composition. The method comprises steps of(a) providing a microorganism that expresses a recombinant pepsinogen;(b) culturing the microorganism under conditions in which therecombinant pepsinogen polypeptide is expressed and secreted by themicroorganism into a growth media; (c) harvesting the growth media andremoving the microorganism therefrom to obtain a liquid startingmaterial; (d) lowering the pH of the liquid starting material to lessthan pH 4.0 to obtain an activated pepsin composition; and (e) raisingthe pH of the activated pepsin composition to a pH of between about 5.4and 7.0 to obtain a high-activity stable pepsin composition.

In some embodiments, the method further comprises, after step (d), astep of isolating activated pepsin polypeptide from other proteins andsmall molecules in the activated pepsin composition and/or, after step(e), a step of isolating activated pepsin polypeptide from otherproteins and small molecules in the high-activity stable pepsincomposition.

Provided herein are methods for producing compositions of recombinantpepsinogen that are stable and can be activated to a high level ofspecific activity.

Another aspect of the present disclosure is a method of producing arecombinant pepsinogen. The method including steps of (a) providing amicroorganism that expresses a recombinant pepsinogen, in which theexpressed pepsinogen is secreted by the microorganism into a growthmedia; (b) culturing the microorganism until the secreted pepsinogen ispresent in the growth medium in an amount of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greaterthan 20 g/liter; and (c) harvesting the growth media and removing themicroorganism therefrom to obtain a liquid material in which therecombinant pepsinogen is substantially in a proteolytically inactiveform.

The methods herein comprise a step of expressing recombinant pepsinogenin a host cell. As used herein, a “host” or “host cell” denotes here anyprotein production host selected or genetically modified to produce adesired product. Exemplary hosts include fungi, such as filamentousfungi, as well as bacteria, yeast, plant, insect, and mammalian cells. Ahost cell may be Arxula spp., Arxula adeninivorans, Kluyveromyces spp.,Kluyveromyces lactis, Komagataella phaffii, Pichia spp., Pichia angusta,Pichia pastoris, Saccharomyces spp., Saccharomyces cerevisiae,Schizosaccharomyces spp., Schizosaccharomyces pombe, Yarrowia spp.,Yarrowia lipolytica, Agaricus spp., Agaricus bisporus, Aspergillus spp.,Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Colletotrichumspp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica,Escherichia coli, Fusarium spp., Fusarium graminearum, Fusarium solani,Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp.,Myceliophthora thermophila, Neurospora spp., Neurospora crassa,Penicillium spp., Penicillium camemberti, Penicillium canescens,Penicillium chrysogenum, Penicillium (Talaromyces) emersonii,Penicillium funiculo sum, Penicillium purpurogenum, Penicilliumroqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp.,Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopusarrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp.,Trichoderma altroviride, Trichoderma reesei, or Trichoderma vireus. Ahost cell can be an organism that is approved as generally regarded assafe by the U.S. Food and Drug Administration.

A host cell may be a microorganism. In some embodiments, themicroorganism is selected from yeast, filamentous fungi, a saccharomycesspecies, a bacteria, a Pichia species, a Trichoderma species and anAspergillus species.

In some embodiments, the host cell for recombinant pepsinogen productioncan be a Pichia species (Komagataella phaffii and Komagataellapastoris), a Saccharomyces species, a Trichoderma species, a Pseudomonasspecies or an E. coli species. In some embodiments, pepsinogen isexpressed in a Pichia species, such as Komagataella phaffii.

The recombinant expression of pepsinogen in a host cell can be regulatedby a promoter.

In some embodiments, the microorganism (or host cell) that expresses therecombinant pepsinogen comprises a first inducible promoter whichregulates the expression of the recombinant pepsinogen. In some methodsof the present disclosure comprise a step of inducing the expression ofrecombinant pepsinogen after or at least partially concurrent with thestep of culturing the microorganism or host cell. In some cases, thefirst inducible promoter is induced by methanol.

Promoters include, but are not limited to, acu-5, adhl+, alcoholdehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, α-amylase,alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2(AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbhl), ccg-1, cDNA1,cellular filament polypeptide (cfp), cpc-2, ctr4+, CUP1,dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehydedehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), G1, G6, GAA,GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14,gdhA, gla-1, α-glucoamylase (glaA), glyceraldehyde-3-phosphatedehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1),glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1),acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, β-galactosidase(lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmtl, NSP, pcbC, PET9,peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), phol, PHO5,PHO89, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase(pki1), RPS7, sorbitol dehydrogenase (SDH), 3-phosphoserineaminotransferase (SERI), SSA4, SV40, TEF, translation elongation factor1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triosephosphate isomerase (TPI1), XRP2, YPT1. In some embodiments of themethods, the expression of pepsinogen is achieved by regulating theexpression using an inducible promoter. Exemplary inducible promotersthat can be used for expression include, but are not limited to methanolinducible promoters, such as alcohol oxidase promoters AOX1 and AOX2,and sugar inducible promoters such as glucose-induced and rhamnoseregulated promoters.

In some embodiments, the recombinant pepsinogen expressed in the hostcell is secreted without conversion to a proteolytically active formsuch that the recombinant pepsinogen is present in and can be isolatedfrom the growth media in which the host cell is grown. Secretion ofrecombinant pepsinogen can be achieved by including a secretion signalin the expression construct, which can be cleaved off as the polypeptideis transited through the host cell secretory pathway. In someembodiments, the secretion signal is present at the N-terminus of therecombinant pepsinogen polypeptide (for example, the bolded sequence inSEQ ID NO: 1). Exemplary secretion signals include but are not limitedto the mating factor α-factor pro sequence from Cerevisiae, an Ostlsignal sequence, hybrid Ost1-α-factor pro sequence, and synthetic signalsequences. In some embodiments, the pepsinogen expression constructsinclude a heterologous secretion signal (e.g., not derived natively frompepsinogen). In some embodiments, the pepsinogen expression constructsinclude a heterologous secretion signal and lack any secretion signalnaturally-derived from or associated with a native pepsinogen codingsequence.

Expression constructs can also include transcriptional terminators.Exemplary transcriptional terminator elements include, but are notlimited to, acu-5, adhl+, alcohol dehydrogenase (ADH1, ADH2, ADH4),AHSB4m, AINV, alcA, α-amylase, alternative oxidase (AOD), alcoholoxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV,cellobiohydrolase I (cbhl), ccg-1, cDNA1, cellular filament polypeptide(cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase(ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formatedehydrogenase (FMDH), G1, G6, GAA, GAL1, GAL2, GAL3, GAL4, GAL5, GAL6,GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, α-glucoamylase (glaA),glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH),phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+,isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5),KAR2, KEX2, β-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase(MOX), nmt1, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase(PGK, PGK1), pho1, PHO5, PHO89, phosphatidylinositol synthase (PIS1),PYK1, pyruvate kinase (pki1), RPS7, sorbitol dehydrogenase (SDH),3-phosphoserine aminotransferase (SER1), SSA4, SV40, TEF, translationelongation factor 1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi,TPS1, triose phosphate isomerase (TPI1), XRP2, and YPT1.

In some embodiments, the microorganism further comprises a helperfactor, e.g., a helper factor that is expressed from a second induciblepromoter. In some cases, the second inducible promoter is induced bymethanol.

Pepsinogen coding sequences for use in producing recombinant pepsinogeninclude animal pepsinogen sequences such as a pig, a sheep, a CentralEuropean red deer, a goat, a cow, a human, a yak, or a zebu pepsinogen.In some embodiments, a pepsinogen sequence is one of SEQ ID NOs: 1-9.The below table also includes the amino acid sequence for an activepepsin (SEQ ID NO: 10).

SEQ ID Sequence name NO Sequence Porcine 1MKWLLLLSLVVLSECLVKVPLVRKKSLRQNLIKNGKLKDFLKTHKHNPASK Pepsinogen (Pre-YFPEAAALIGDEPLENYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVY pro form ofCSSLACSDHNQFNPDDSSTFEATSQELSITYGTGSMTGILGYDTVQVGGIS Pepsin)DTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISASGATPVFDNLWDQGLVSQDLFSVYLSSNDDSGSVVLLGGIDSSYYTGSLNWVPVSVEGYWQITLDSITMDGETIACSGGCQAIVDTGTSLLTGPTSAIANIQSDIGASENSDGEMVISCSSIDSLPDIVFTINGVQYPLSPSAYILQDDDSCTSGFEGMDVPTSSGELWILGDVFIRQYYTVFDRANNKVGLAPVA Pepsinogen 2EAEALVKVPLVRKKSLRQNLIKNGKLKDFLKTHKHNPASKYFPEAAALIGDE sequence (byPLENYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVYCSSLACSDHNQF EdmanNPDDSSTFEATSQELSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEP sequencing)GSFLYYAPFDGILGLAYPSISASGATPVFDNLWDQGLVSQDLFSVYLSSNDDSGSVVLLGGIDSSYYTGSLNWVPVSVEGYWQITLDSITMDGETIACSGGCQAIVDTGTSLLTGPTSAIANIQSDIGASENSDGEMVISCSSIDSLPDIVFTINGVQYPLSPSAYILQDDDSCTSGFEGMDVPTSSGELWILGDVFIRQYYTVFD RANNKVGLAPVAOvis cries (sheep) 3MKWLLLLALVVLSECSVFKIPLVKKKSLRQNLIENGKLKEFMKTHKYNLGSK PepsinogenYIREAATLVSDQPLQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSIYCSSEACTNHNRFNPQDSSTYEATSETLSITYGTGSMTGILGYDTVEVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWDQGLVSQDLFSVYLSSNEESGSVVMFGGIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEEVISCSSIDSLPDIVFTINGVQYPVPPSAYILQNDDVCSSGFEGMDIPTSSGDLWILGDVFIRQYFTVFDRANNQIGLAPVA Cervus elaphus 4MLRHRIPLVKKKSLRRNLIENGKLKEFMQTHKYNLASKYFPETATLVSDQPL hippelaphusQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSIYCSSEACTNHNRFN (central EuropeanPEDSSTYEATSETLSITYGTGSMTGILGYDTVQVGGITDTNQIFGLSETEPGS red deer)FLYYAPFDGILGLAYPSISSSGATPVFDNIWDQGLVSQDLFSVYLSSNEESG PepsinogenSVVIFGDIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEVVISCSSIDSLPDVVFTINGVQYPVPPSAYILQSDGVCSSGFEGMDVSTSSGDLWILGDVFIRQYYTVFDRAN NQIGLAPVACapra hircus 5 MKWLLLLALVVLSECSFFKIPLVKKKSLRQNLIENGKLKEFMKTHKYNLGSK(Goat) YIREAATLVSDQPLQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVY PepsinogenCSSEACTNHNRFNPQDSSTYEATSETLSITYGTGSMTGVLGYDTVEVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWDQGLVSQDLFSVYLSSNEESGSVVIFGGIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEEVISCSSIDSLPDIVFTINGVQYPVPPSAYILQSDDVCSSGFEGMDISTSSGDLWILGDVFIRQYFTVFDRANNQIGLAPVA Bos taurus 6MKWLLLLALVALSECSVVKIPLVKKKSLRQNLIENGKLKEFMRTHKYNLGSK (Bovine)YIREAATLVSEQPLQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSIYC PepsinogenSSEACTNHNRFNPQDSSTYEATSETLSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWDQGLVSQDLFSVYLSSNEESGSVVIFGDIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEVVISCSSIDSLPDIVFTINGVQYPVPPSAYILQSNGICSSGFEGMDISTSSGDLWILGDVFIRQYFTVFDRGNNQIGLAPVA Homo sapiens 7MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNPARK PepsinogenYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGSSNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQDLFSVYLSADDKSGSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGETIACAEGCQAIVDTGTSLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEGSCISGFQGMNVPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA Bos mutus (yak) 8RIMKWLLLLALVALSECSVVKIPLVKKKSLRQNLIENGKLKEFMRTHKYNLG PepsinogenSKYIREAATLVSEQPLQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSIYCSSEACTNHNRFNPQDSSTYEATSETLSITYGTGSMTGVLGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWDQGLVSQDLFSVYLSSNEESGSVVIFGDIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEVVISCSSIDSLPDIVFTINGVQYPVPPSAYILQSDGICSSGFEGMDISTSSGDLWILGDVFIRQYFTVFDRGNNQIGLAPVA Bos indicus (Zebu) 9MKWLLLLALVALSECSVVKIPLVKKKSLRQNLIENGKLKEFMRTHKYNLGSK PepsinogenYIREAATLVSEQPLQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSIYCSSEACTNHNRFNPQDSSTYEATSETLSITYGTGSMTGVLGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSRATPVFDNIWDQGLVSQDLFSVYLSSNEESGSVVIFGDIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEVVISCSSIDSLPDIVFTINGVQYPVPPSAYILQSDGICSSGLEGMDISTSSGDLWILGDVF1RQYFTVFDRGNNQIGLAPVA Pepsin sequence 10IGDEPLENYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVYCSSLACSD (by EdmanHNQFNPDDSSTFEATSQELSITYGTGSMTGILGYDTVQVGGISDTNQIFGL sequencing,SETEPGSFLYYAPFDGILGLAYPSISASGATPVFDNLWDQGLVSQDLFSVYLSSNDDSGSVVLLGGIDSSYYTGSLNWVPVSVEGYWQITLDSITMDGETIACSGGCQAIVDTGTSLLTGPTSAIANIQSDIGASENSDGEMVISCSSIDSLPDIVFTINGVQYPLSPSAYILQDDDSCTSGFEGMDVPTSSGELWILGDVFIRQYY TVFDRANNKVGLAPVA

A recombinant pepsinogen or recombinant pepsin can include additionalsequences. Expression of recombinant pepsinogen or recombinant pepsin ina host cell, for instance a Pichia species, a Saccharomyces species, aTrichoderma species, a Pseudomonas species may lead to an addition ofpeptides to the pepsinogen or pepsin sequence as part ofpost-transcriptional or post-translational modifications. Such peptidesmay not be part of the native pepsinogen or pepsin sequences. Forinstance, expressing a pepsinogen sequence in a Pichia species, such asKomagataella phaffii and Komagataella pastoris may lead to addition of apeptide at the N-terminus or C-terminus. In some cases, a tetrapeptideEAEA (SEQ ID NO: 11) is added to the N-terminus of the pepsinogensequence upon expression in a host cell. In some embodiments, pepsinogenor pepsin or both include the amino acids EAEA (SEQ ID NO: 11) at theN-terminus for example, in SEQ ID NO: 2.

A recombinant pepsinogen polypeptide can be a non-naturally occurringvariant of a pepsinogen. Such a variant can comprise one or more aminoacid insertions, deletions, or substitutions relative to a nativepepsinogen sequence.

Similarly, a recombinant pepsin polypeptide can be a non-naturallyoccurring variant of a pepsin. Such a variant can comprise one or moreamino acid insertions, deletions, or substitutions relative to a nativepepsin sequence. Variants of pepsinogen can have at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs:1-9 and variants of pepsin can have at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. The term“sequence identity” as used herein in the context of amino acidsequences is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues in aselected sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full-length of the sequences being compared.

In some embodiments, a variant is one that confers additional features,such as reduced allergenicity. Depending on the host organism used toexpress the recombinant pepsinogen, it can have a glycosylation,acetylation, or phosphorylation pattern different from wildtypepepsinogen and/or wildtype pepsin. For example, the recombinantpepsinogen or recombinant pepsin disclosed herein may or may not beglycosylated, acetylated, or phosphorylated. A recombinant pepsinogen ora recombinant pepsin may have an avian, non-avian, microbial,non-microbial, mammalian, or non-mammalian glycosylation, acetylation,or phosphorylation pattern.

In some cases, recombinant pepsinogen or recombinant pepsin may bedeglycosylated (e.g., chemically, enzymatically, Endo-H, PNGase F,O-Glycosidase, Neuraminidase, β1-4 Galactosidase,β-N-acetylglucosaminidase), deacetylated (e.g., protein deacetylase,histone deacetylase, sirtuin), or dephosphorylated (e.g., acidphosphatase, lambda protein phosphatase, calf intestinal phosphatase,alkaline phosphatase). Deglycosylation, deacetylation ordephosphorylation may produce a polypeptide that is more uniform or iscapable of producing a composition with less variation.

The pepsinogen expression constructs and host cells (e.g., amicroorganism) can be used to produce recombinant pepsinogen in liquidculture, such as in a test tube, shaker flask, or small-scale andlarge-scale fermentation vessel. In the methods provided herein, thehost cell carrying the pepsinogen expression construct can be initiallycultured under conditions where there is little to no expression ofpepsinogen as a starter culture, and grown to a target cell number,density or for a target duration (referred to as a “growth phase”).

In some embodiments of the method, after such growth phase, recombinantpepsinogen expression can be initiated (“expression phase”). In someembodiments, expression is initiated such as by induction of aninducible promoter, e.g., a promoter induced by methanol. In someembodiments, expression is initiated such as by release of a repressiblepromoter or by removal of a blocking sequence, protein binding or otherform of repression of expression. In other cases, the expression ofpepsinogen can be driven by a constitutive promoter.

In some embodiments, the pH of the culture media is controlled duringthe growth phase, the expression phase, or during both phases. In someembodiments, the pH of the growth phase is about pH 5. In someembodiments, the pH of the growth media is about 5, and then isincreased to about pH 6 before the expression phase.

After initiation of the expression phase, the culturing is continued fora target length of time or up until a target amount of recombinantpepsinogen is recovered from the culture media. In the methods disclosedherein, the cultured host organism can provide a titer of at least about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20g/L of total protein or greater than 20 g/liter.

By producing pepsinogen recombinantly, the ultimate recombinant pepsincomposition will comprise fewer additional and contaminating proteins,for example when compared to extracting pepsin from natural sources,e.g. from a pig's digestive system. In particular, when extractingpepsin from natural sources, contaminating animal proteins will beincluded in the extract. In embodiments, a recombinant pepsincomposition may comprise less than 5% of impurities/contaminatingproteins, i.e., non-pepsin proteins. For example, a recombinant pepsincomposition may comprise less than 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%,1.5%, 1.0%, 0.5% or less of contaminating proteins. In variousembodiments, a recombinant pepsin composition comprises little to nocontaminating animal proteins. In some embodiments, a recombinant pepsincomposition is free from animal-derived proteins.

In some embodiments, the method further comprises steps of isolatingpepsinogen and/or activated pepsin polypeptide from other proteins andsmall molecules present in a growth medium, a liquid starting medium,and/or a composition.

Recombinant pepsin and/or recombinant pepsinogen compositions of thepresent disclosure may include additional proteins, e.g., proteins thatare added to promote desirable qualities and features to a composition.

Pepsinogen Protein Isolation and Conversion to Pepsin

The methods herein can include one or more steps whereby the recombinantpepsinogen is separated from the host cell and other culture mediacomponents. Host cells, some host cell proteins, and cellular debris canbe removed through centrifugation, filtration or a combination thereof.However, such gross separation of the host cell and other culture mediacomponents does not result in a purified recombinant pepsinogencomposition and/or an isolated recombinant pepsinogen composition asadditional proteins and other molecules remain in the composition thatcontains the pepsinogen.

In some embodiments, the growth medium is filtered and/or centrifugedafter culturing a microorganism, a liquid starting material is filteredand/or centrifuged after harvesting a growth medium, and/or a final orpenultimate composition is filtered and/or centrifuged.

In some embodiments, a composition comprises recombinant pepsinogen inits stable zymogen (inactive) form that can be activated under specificconditions. Such compositions provide improved stability and control ofactivity upon conversion to a proteolytically active form as compared tothe proteolytically active enzyme extracted from animal gut and othersources.

In some embodiments, a composition contains recombinant pepsinogen in astable, inactive form and the compositions are substantially recombinantpepsinogen and contain low amounts or little to no recombinant pepsin.In some embodiments, the compositions have a ratio of recombinantpepsinogen to recombinant pepsin of at least about 10:1, 100:1, 1000:1or greater than 1000:1.

In some embodiments, methods for producing a composition of the presentdisclosure include one or more pH shift steps to convert recombinantpepsinogen to recombinant pepsin and to maintain recombinant pepsin in astable form. The recombinant pepsinogen compositions disclosed hereincan be activated to convert the recombinant pepsinogen to the pepsinform of the enzyme, such as by lowering the pH of the composition, byaddition of an acidic ingredient, addition of acid, or by placing thecomposition in an acidic environment. In some embodiments, in a first pHshift step, the pH can be lowered below pH 5, such as to about pH 4 orpH 3.5 or pH 2.0 for a period of time to convert recombinant pepsinogento recombinant pepsin. Such a pH shift can be performed at roomtemperature or at about 20° C. to about 25° C. or at a temperature at orabout 10° C.-45° C. In some embodiments, the duration for treating at apH below 5, e.g., about pH 4 or about pH 3.5, is about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In someembodiments, the duration for treating at a pH below 5, e.g., about pH 4or about pH 3.5, is about 1 hour, 2 hours, 2.25 hours, or 2.5 hours.

In embodiments, growth media comprising recombinant pepsinogen isharvested and cellular material is removed, thereby obtaining a liquidstarting material. The pH of the liquid starting material is lowered toless than pH 4.0 (such as pH 3.5) to obtain an activated recombinantpepsin composition. The pH may be lowered by the addition of an acid,for example hydrochloric acid, phosphoric acid, sulfuric acid, or nitricacid. Thus, in embodiments, it is unnecessary to first purify therecombinant pepsinogen, e.g., from a growth media, before converting therecombinant pepsinogen to recombinant pepsin. Surprisingly, a purerrecombinant pepsin product which has higher activity is provided byconverting newly-synthesized recombinant pepsinogen to recombinantpepsin and subsequently purifying the recombinant pepsin rather thanpurifying the recombinant pepsinogen.

In embodiments, the compositions are composed of primarily recombinantpepsin and have little or no detectable pepsinogen or otherintermediates derived from pepsinogen. In some embodiments, the amountof recombinant pepsinogen in the composition comprising recombinantpepsin is less than about 10%, 5%, 1%, 0.5%, 0.1% or 0.05% (weightpepsinogen/weight pepsin).

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsin.

In some embodiments, the composition comprises a protein content of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% w/w.

In some embodiments, active recombinant pepsin is then converted tostable (e.g., inactive) recombinant pepsin, such as by raising the pH.In some embodiments, a second pH shift step raises the pH to at leasthas a pH 5.4, e.g., about pH 6 or above pH 6, following the first pHshift step to maintain pepsin in a stable form that is enzymaticallyinactive when held at such pH conditions.

Raising the pH of a pepsin composition to greater than 5.4, e.g., aboutpH 6.0, obtains a stable recombinant pepsin composition. By stable ismeant, in part, that the recombinant pepsin does not substantiallydigest itself and remains in an intact form. The compositions of thepresent disclosure comprise recombinant pepsins that are intact and in astable proteolytically inactive form. This form is not present innatural pepsin because natural pepsin compositions self-digest over time(i.e., they are not stable in maintaining a substantially intact form ofpepsin in the composition). Such an intact and stable proteolyticallyinactive form of the recombinant pepsin compositions provided hereinadvantageously allows long-term storage (at room temperature or atrefrigeration temperature) of compositions of the present disclosure.Long-term storage may be for a month, two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months or longer. After suchlong-term storage, the recombinant pepsin can be activated by loweringthe pH of a composition. Enzymic activity of the re-activatedrecombinant pepsin is maintained after long-term storage, e.g., withinabout 15% of a composition's original activity.

In some embodiments, following the pH shift steps, the stablerecombinant pepsin can be purified. For example, by removingcontaminating proteins, carbohydrates, lipids, salts, nucleic acids,small molecules, cells (e.g., microorganism), or cell components (e.g.,cellular debris). In embodiments which include a step of “harvesting thegrowth media and removing the microorganism therefrom”, the removal ofthe microorganism may be a consequence of harvesting the growth mediumand not a separate step of “removing”. For example, the growth mediummay be filtered and/or centrifuged during harvesting; either of thesewould remove microorganisms in the growth medium. Alternately, “removingthe microorganism” may be a distinct step from the harvesting the growthmedium; this distinct step purposefully removes the microorganism.

In some embodiments, following the pH shift steps, the stable pepsin canbe concentrated, such as to a 2×, 3×, 4×, 5×, 10× or greater than 10×concentrated form. In some embodiments, the concentrate is maintained asa liquid. In some embodiments, the concentrate is lyophilized or driedand stored as a solid or powder. In some embodiments, the stable pepsinconcentrate can be diluted for formulation, for final product productionor for consumption. In some embodiments, the diluted stable pepsin ismaintained at a pH to about pH 6 (e.g., 5.4 to 6.0) or above pH 6. Insome embodiments, the diluent for the pepsinogen composition is NaCl.

In some embodiments, stable recombinant pepsin is subsequently convertedto an enzymatically active form, such as by lowering the pH (“activatedrecombinant pepsin composition”). Activated recombinant pepsincompositions provided herein can have a high specific activity.

In some embodiments, the method further comprises a step of isolatingthe activated pepsin polypeptide from contaminating proteins,carbohydrates, lipids, salts, nucleic acids, and small molecules in theliquid starting material and the lowering of the pH of the liquidstarting material may be performed after the isolating the recombinantpepsin polypeptide to provide an activated recombinant pepsincomposition. In some embodiments, the isolating of the activated pepsinpolypeptide occurs after a step of lowering the pH of the liquidstarting material to less than pH 4.0 to obtain an activated pepsincomposition and a step of raising the activated pepsin composition to apH of about 6.0 to obtain a high-activity stable pepsin composition.

For example, a stable and inactive recombinant pepsin composition, whichhas a pH of about 6 (e.g., between 5.4 and 6.0), can be converted intoan active recombinant pepsin composition by lowering the pH of thecomposition to less than pH 4.0.

In some embodiments, a method further comprises a desalting step after aharvesting pepsinogen or pepsin and/or the method may further comprise adrying step after the harvesting. The drying step may be spray drying orlyophilization.

Compositions and Uses of Compositions

Recombinant pepsin compositions can include stable recombinant pepsin, aconcentrate of stable recombinant pepsin, a dilution of stablerecombinant pepsin, as well as mixtures of stable recombinant pepsinwith one or more additional ingredients. Recombinant pepsin compositionsof the present disclosure may include additional proteins, e.g.,proteins added to promote desirable qualities and features to acomposition.

In an aspect, the present disclosure provides a composition comprising arecombinant pepsin polypeptide. The composition is (a) free fromanimal-derived proteins, (b) the pepsin polypeptide is substantially inan intact and stable proteolytically inactive form, (c) the compositionhas a pH greater than about 5.4, and (d) the composition has a specificactivity of at least 20,000 FCC units/mg total protein.

In some embodiments, a composition comprising recombinant pepsin issubstantially free from animal-derived proteins.

Recombinant pepsin compositions also can include activated recombinantpepsin compositions, a dilution of activated recombinant pepsincompositions, as well as mixtures of activated recombinant pepsincompositions with one or more additional ingredients. Recombinant pepsincompositions of the present disclosure may include additional proteins,e.g., proteins added to promote desirable qualities and features to acomposition.

In some embodiments, the recombinant pepsin compositions aresubstantially free contaminating proteins, carbohydrates, lipids, salts,nucleic acids, and small molecules.

In some embodiments, the activated recombinant pepsin compositionsdisclosed herein (after the pH has been lowered) provide a specificactivity that is higher than commercially-available pepsin-relatedproducts. In some embodiments, the activated recombinant pepsincomposition provided herein have a specific activity that is at leastabout 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90% 100%, 150%,200%, or 250% higher than a commercially-available pepsin-relatedproduct. In some embodiments, the activated recombinant pepsincomposition provided herein have a specific activity that is at leastabout 1.2, 1.5, 1.7, 2, 2.5, 3, 3.5 or greater than 3.5-fold higher thana commercially-available pepsin-related product.

In some embodiments, a recombinant pepsin composition, such as arecombinant pepsin composition comprising a pepsin polypeptidesubstantially in an intact and stable proteolytically inactive form, isin a powdered form of the composition. In some embodiments, arecombinant pepsin composition, such as a recombinant pepsin compositioncomprising a pepsin polypeptide substantially in an intact and stableproteolytically inactive form, is in a liquid form of the composition.The proteolytically inactive pepsin polypeptide form (in a powdered or aliquid composition) may be stable for at least six months at roomtemperature, e.g., six months, seven months, eight months, nine months,ten months, eleven months, twelve months or longer. The proteolyticallyinactive pepsin polypeptide form (in a powdered or a liquid composition)may be stable in the composition for at least six months at 4° C., e.g.,six months, seven months, eight months, nine months, ten months, elevenmonths, twelve months or longer. A powdered composition may have amoisture content of less than about 10%, e.g., less than 10%, less than9%, less than 8%, less than 7%, less than 6%, less than 5%, or lower. Insome embodiments, a composition comprises a protein content of at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%w/w; the remaining substances in a composition may be additional organicmatter.

In embodiments, the compositions are composed of primarily recombinantpepsin and have little or no detectable pepsinogen or otherintermediates derived from pepsinogen. In some embodiments, the amountof recombinant pepsinogen in the composition comprising recombinantpepsin is less than about 10%, 5%, 1%, 0.5%, 0.1% or 0.05% (weightpepsinogen/weight pepsin).

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsin.

In some embodiments, a recombinant pepsin composition has a specificactivity at pH of 2 of at least 20,000 FCC units/mg total protein(expressed as units of pepsin activity per total of all protein in thecomposition), e.g., of at least 25,000 FCC units/mg total protein, of atleast 30,000 FCC units/mg total protein, of at least 35,000 FCC units/mgtotal protein, at least 40,000 FCC units/mg total protein, of at least45,000 FCC units/mg total protein, at least 50,000 FCC units/mg totalprotein, of at least 55,000 FCC units/mg total protein, at least 60,000FCC units/mg total protein, of at least 65,000 FCC units/mg totalprotein, and at least 70,000 FCC units/mg total protein. The FCCunits/mg total protein may relate to the total amount of recombinantpepsin protein alone. As mentioned above, a recombinant pepsincompositions of the present disclosure may include additional proteins;thus, the FCC units/mg total protein may relate to the recombinantpepsin protein and the additional proteins. An FCC unit (also referredto herein as a pepsin unit) is defined as that quantity of enzyme thatproduces the equivalent of 1 μmol of tyrosine per min under theconditions of incubating the enzyme with 2% hemoglobin substrate at pH1.6 for 10 minutes at 25° C. (see Food Chemical Codex, 11th ed.(Pharmacopeial Convention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, a powdered recombinant pepsin composition has aspecific activity at pH of 2 of at least 20,000 FCC units/mg totalpowder, e.g., of at least 25,000 FCC units/mg total powder, of at least30,000 FCC units/mg total powder, of at least 35,000 FCC units/mg totalpowder, at least 40,000 FCC units/mg total powder, at least 45,000 FCCunits/mg total powder, at least 50,000 FCC units/mg total powder, atleast 55,000 FCC units/mg total powder, at least 60,000 FCC units/mgtotal powder, at least 65,000 FCC units/mg total powder, and at least70,000 FCC units/mg total powder. As used herein, an FCC unit (alsoreferred to herein as a pepsin unit) is defined as that quantity ofenzyme that produces the equivalent of 1 μmol of tyrosine per min underthe conditions of incubating the enzyme with 2% hemoglobin substrate atpH 1.6 for 10 minutes at 25° C. (see Food Chemical Codex, 11th ed.(Pharmacopeial Convention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, the recombinant pepsin polypeptide in a compositioncomprises SEQ ID NO: 10, or an amino acid sequence with at least 90%identity thereto.

In another aspect, the present disclosure provides a compositioncomprising a recombinant pepsinogen polypeptide. The composition is (a)free from animal-derived proteins, (b) the pepsinogen polypeptide issubstantially in an intact and stable proteolytically inactive form, (c)the composition has a pH greater than about 5.4, and (d) the pepsinogenpolypeptide is capable of being activated to a proteolytically activepepsin form that has a specific activity of at least 20,000 FCC units/mgtotal protein.

Recombinant pepsinogen compositions can include recombinant pepsinogen,a concentrate of recombinant pepsinogen, as well as mixtures ofrecombinant pepsinogen with one or more additional ingredients.

In some embodiments, the recombinant pepsinogen compositions disclosedherein, have a significantly lower amount of pepsin in the compositioninitially, e.g., such as when secreted from a host cell, as compared tocommercially-available pepsin-related products. In some embodiments, theamount of recombinant pepsin in the recombinant pepsinogen compositionsdisclosed herein is less than 50%, 25%, 10%, 5%, 1%, 0.1% or less than0.1% of the pepsin found in commercially-available pepsin-relatedproducts. In some cases, the amount of pepsin in a recombinantpepsinogen composition is less than 10%, 5%, 1%, 0.5%, 0.1% or 0.05%(weight pepsin/weight pepsinogen). The formulated composition may besubstantially devoid of pepsin.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsinogen.

In some embodiments, the composition comprises a protein content of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% w/w.

In some embodiments, the recombinant pepsinogen in a compositioncomprises an amino acid sequence of one of SEQ ID Nos. 1-9 or a sequencewith at least 90% identity thereto.

A consumable composition can be an ingredient of a final product orfinished product. For example, recombinant pepsinogen or recombinantpepsin can be mixed with water or other liquids to form aproteolytically inactive solution of recombinant pepsinogen orrecombinant pepsin. In some cases, recombinant pepsinogen or recombinantpepsin can be mixed with water or other liquids to form aproteolytically active solution of recombinant pepsin. This solution canbe an ingredient that is then mixed with other ingredients to make afinal product for an end-user. A final or finished product is one thatis ready for an end-user's use, such as for use in a food-making orindustrial process, or for use as a digestive aid or treatment by anend-user for consumption by an animal, such as a human, companion animalor livestock. The finished product can be a processed product, such asprocessed food or a processed drink, or an industrial product, such aspreparation of proteins, antibodies and peptides for use in medicine andin uses such as hide and leather preparation. In some instances, thepepsinogen or pepsin is provided in a separate container to be mixedinto the final product or with other components to make a final productby the end-user.

In some embodiments, the recombinant pepsinogen compositions andrecombinant pepsin compositions provided herein are formulated.Formulation can include ingredients suitable to create an orallyconsumable ingredient or orally administered pharmaceutical formulation.A formulated comprising recombinant pepsinogen or recombinant pepsin maycomprise at least 2 g, 5 g, 7 g, 10 g, 15 g, 20 g of the enzyme perliter of the composition.

In some embodiments, a recombinant pepsinogen or recombinant pepsincompositions disclosed herein are formulated with at least oneingredient to form a digestive aid, such as in a pill, powder, tablet,capsule, caplets, liquid, syrup, gel or other suitable forms for humanand animal oral ingestion. A recombinant pepsinogen composition or arecombinant pepsin composition may be formulated as a microencapsulateor liposomes suspended in syrups, liquids, sugar and pectin-basedconfectionary. Digestive aid recombinant pepsinogen or recombinantpepsin compositions can be ingested by an animal, including but notlimited to human, companion animal or farm animal, to provide pepsin toaid the animal's digestion. For example, such digestive aid recombinantpepsin composition can be taken orally with the form of the enzyme inthe composition as primarily or substantially the inactive pepsin form.Upon reaching an acidic environment in the animal's gut, the pepsin isconverted into the active enzymatic form of pepsin and then therecombinant pepsin can aid in breaking down other proteins in theanimal's gut to aid in the animal's digestion and improve nutrientabsorption.

The at least one ingredient may be a food additive orpharmaceutically-acceptable excipient. These ingredients can add volumeand/or mass to a composition. They may improve functional performanceand/or physical characteristics. The ingredient may relate to pHadjustment; examples include, but are not limited to, Tris buffer,potassium phosphate, sodium hydroxide, potassium hydroxide, citric acid,sodium citrate, sodium bicarbonate, and hydrochloric acid. Theingredient may be a salt; examples include, but are not limited, to acidsalts, alkali salts, organic salts, inorganic salts, phosphates,chloride salts, sodium salts, sodium chloride, potassium salts,potassium chloride, magnesium salts, magnesium chloride, magnesiumperchlorate, calcium salts, calcium chloride, ammonium chloride, ironsalts, iron chlorides, zinc salts, and zinc chloride. The ingredient maybe a carbohydrate; examples include, but are not limited to, sugar,sucrose, glucose, fructose, galactose, lactose, maltose, mannose,allulose, tagatose, xylose, arabinose, high fructose corn syrup, highmaltose corn syrup, corn syrup (e.g., glucose-free corn syrup), sialicacid, monosaccharides, disaccharides, and polysaccharides (e.g.,polydextrose, maltodextrin). The ingredient may be a gum; examplesinclude, but are not limited to, gum arabic, gellan gum, guar gum,locust bean gum, acacia gum, cellulose gum, and xanthan gum. Theingredient may be a liquid; examples include, but are not limited water,an aqueous buffer, an oil, a gel, and a carbohydrate-containing syrup orfluid.

In some embodiments, a digestive aid recombinant pepsin composition ismixed with an ingredient, such as lactose, to modify its enzymaticactivity.

In some embodiments, the recombinant pepsinogen or recombinant pepsincompositions disclosed herein are used for preparation of food, beverageand other consumable compositions, such as for products that have soy orgelatin as ingredients. Recombinant pepsinogen or recombinant pepsincompositions disclosed herein can be used for making animal andvegetable protein hydrolysates for use in flavoring foods and beverages,and for making snack items and instant hot cereals. Exemplary usesinclude but are not limited to ale, beer, light beer, malt liquor,porter, stout, cheese (such as cheddar, cottage cheese, cream cheese,cream cheese spread), defatted soya flour, pre-cooked instant breakfastcereals, and hydrolyzed animal, milk and vegetable proteins. Recombinantpepsinogen or recombinant pepsin compositions also have utility intreating allergen-causing food items, such as legumes, to reduceallergic reactions when consumed by an animal, such as for humanconsumption.

In some embodiments, the recombinant pepsinogen or recombinant pepsincompositions disclosed herein are useful for preparation of biologicaltools and therapeutics. For example, the recombinant pepsinogen orrecombinant pepsin compositions can be converted to compositioncontaining active pepsin and employed to create antibody fragments, suchas Fabs, that can be used for diagnostic and therapeutic applications,as well as used as tools in biotechnology.

A recombinant pepsinogen composition or a recombinant pepsin compositionmay also be formulated for the treatment of disease or condition of thegastrointestinal tract. Such a method may include steps of (a) providingthe recombinant pepsinogen in a formulated composition, in which thepepsinogen is produced by the method according to any herein disclosedmethod and (b) administering the formulated composition for oraladministration; in which upon contact of the formulated composition withan animal gut environment, the pepsinogen is converted to high-activitystable pepsin; and in which the pepsin is effective to treat the diseaseor condition of the gastrointestinal tract. Alternately, a recombinantpepsin composition, as disclosed herein, may be administered to asubject with a disease or condition of the gastrointestinal tract; here,the recombinant pepsin is in a primarily or substantially inactive form.The recombinant pepsin composition may then be activated in thesubject's gastrointestinal tract and release the active form of thepepsin enzyme. A subject can be an animal, such as a human, a companionanimal or livestock animal.

The recombinant pepsinogen or recombinant pepsin compositions disclosedherein can used to make animal-free products and ingredients, such asanimal free pharmaceuticals, digestive aids, food and beverageingredients, food and beverage products and enzyme preparations (such asanimal-free rennet for use in cheese-making.

The recombinant pepsinogen or recombinant pepsin compositions disclosedherein can used for vegetarian, vegan, kosher and halal ingredients andproducts.

Additional Compositions and Methods

An aspect of the present disclosure is a method of producing a stablepepsin composition. The method comprises steps of (a) providing amicroorganism that expresses a recombinant pepsinogen; (b) culturing themicroorganism under conditions in which the recombinant pepsinogenpolypeptide is expressed and secreted by the microorganism into a growthmedia; (c) harvesting the growth media and removing the microorganismtherefrom to obtain a liquid starting material; (d) lowering the pH ofthe liquid starting material to less than pH 4.0 to obtain an activatedpepsin composition; and (e) raising the pH of the activated pepsincomposition to a pH of between about 5.4 and 7.0 to obtain a stablepepsin composition. The stable pepsin composition has a specificactivity at pH of 2 of between about 100 FCC units/mg total protein andabout 19,000 FCC units/mg total protein.

In some embodiments, the method further comprises, after step (d), astep of isolating activated pepsin polypeptide from other proteins andsmall molecules in the activated pepsin composition and/or, after step(e), a step of isolating activated pepsin polypeptide from otherproteins and small molecules in the stable pepsin composition.

In some embodiments, the stable pepsin composition comprises an intactand stable proteolytically inactive form of recombinant pepsin.

In some embodiments, the specific activity at pH of 2 is at least 500FCC units/mg total protein, at least 1000 FCC units/mg total protein, atleast 2000 FCC units/mg total protein, at least 5000 FCC units/mg totalprotein, at least 10000 FCC units/mg total protein, or at least 15000FCC units/mg total protein, and less than 20000 FCC units/mg totalprotein. As examples, the specific activity at pH of 2 is at least 100FCC units/mg total protein, at least 200 FCC units/mg total protein, atleast 300 FCC units/mg total protein, at least 400 FCC units/mg totalprotein, at least 500 FCC units/mg total protein, at least 600 FCCunits/mg total protein, at least 700 FCC units/mg total protein, atleast 800 FCC units/mg total protein, at least 900 FCC units/mg totalprotein, at least 1000 FCC units/mg total protein, at least 1100 FCCunits/mg total protein, at least 1200 FCC units/mg total protein, atleast 1300 FCC units/mg total protein, at least 1400 FCC units/mg totalprotein, at least 1500 FCC units/mg total protein, at least 1600 FCCunits/mg total protein, at least 1700 FCC units/mg total protein, atleast 1800 FCC units/mg total protein, at least 1900 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 3000 FCCunits/mg total protein, at least 4000 FCC units/mg total protein, atleast 5000 FCC units/mg total protein, at least 6000 FCC units/mg totalprotein, at least 7000 FCC units/mg total protein, at least 8000 FCCunits/mg total protein, at least 9000 FCC units/mg total protein, atleast 10000 FCC units/mg total protein, at least 11000 FCC units/mgtotal protein, at least 12000 FCC units/mg total protein, at least 13000FCC units/mg total protein, at least 14000 FCC units/mg total protein,at least 15000 FCC units/mg total protein, at least 16000 FCC units/mgtotal protein, at least 17000 FCC units/mg total protein, at least 18000FCC units/mg total protein, or at least 19000 FCC units/mg total proteinand less than 20000 FCC units/mg total protein. As used herein, an FCCunit (also referred to herein as a pepsin unit) is defined as thatquantity of enzyme that produces the equivalent of 1 μmol of tyrosineper min under the conditions of incubating the enzyme with 2% hemoglobinsubstrate at pH 1.6 for 10 minutes at 25° C. (see Food Chemical Codex,11th ed. (Pharmacopeial Convention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, the final pH of the stable pepsin composition isbetween about 5.4 and 6.0.

In some embodiments, the recombinant pepsinogen is present in at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or greater than 20 g/liter in the growth medium prior to step (c).

In some embodiments, the growth medium is filtered after step (b), theliquid starting material is filtered after step (c), the activatedpepsin composition is filtered after step (d), and/or the stable pepsincomposition is filtered after step (e).

In some embodiments, a recombinant pepsin in the stable pepsincomposition comprises an amino acid sequence of a pig, a sheep, aCentral European red deer, a goat, a cow, a human, a yak, or a zebupepsin.

Another aspect of the present disclosure is a method of producing arecombinant pepsinogen. The method comprises steps of (a) providing amicroorganism that expresses a recombinant pepsinogen, wherein theexpressed pepsinogen is secreted by the microorganism into a growthmedia; (b) culturing the microorganism until the secreted pepsinogen ispresent in the growth medium in an amount of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greaterthan 20 g/liter; and (c) harvesting the growth media and removing themicroorganism therefrom to obtain a liquid material in which therecombinant pepsinogen is substantially in a proteolytically inactiveform. The recombinant pepsinogen that is substantially in theproteolytically inactive form is capable of being activated by reducingthe pH or by exposure to an acidic environment to produce a stablepepsin composition, wherein the stable pepsin composition has a specificactivity at pH of 2 of between about 100 FCC units/mg total protein andabout 19,000 FCC units/mg total protein.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of a pig, a sheep, a Central European red deer, a goat, a cow,a human, a yak, or a zebu pepsinogen.

In some embodiments, the recombinant pepsinogen comprises an amino acidsequence of one of SEQ ID Nos. 1-9 or a sequence with at least 90%identity thereto.

In some embodiments, the microorganism that expresses the recombinantpepsinogen comprises a first inducible promoter which regulates theexpression of the recombinant pepsinogen. In some embodiments, the themethod further comprises a step of inducing the expression of therecombinant pepsinogen after or at least partially concurrent with thestep of culturing the microorganism.

In some embodiments, the microorganism is selected from yeast,filamentous fungi, a saccharomyces species, a bacteria, a Pichiaspecies, a Trichoderma species and an Aspergillus species. In someembodiments, the microorganism is a Pichia species. In some embodiments,the microorganism further comprises a helper factor. In someembodiments, the helper factor is expressed from a second induciblepromoter. In some embodiments, the first inducible promoter, the secondinducible promoter, or both the first and second inducible promoters areinduced by methanol.

In some embodiments, the method further comprises a desalting step afterthe harvesting step and/or a drying step after the harvesting step. Insome embodiments, the drying step comprises spray drying orlyophilization.

In some embodiments, the specific activity at pH of 2 is at least 500FCC units/mg total protein, at least 1000 FCC units/mg total protein, atleast 2000 FCC units/mg total protein, at least 5000 FCC units/mg totalprotein, at least 10000 FCC units/mg total protein, or at least 15000FCC units/mg total protein, and less than 20000 FCC units/mg totalprotein. As examples, the specific activity at pH of 2 is at least 100FCC units/mg total protein, at least 200 FCC units/mg total protein, atleast 300 FCC units/mg total protein, at least 400 FCC units/mg totalprotein, at least 500 FCC units/mg total protein, at least 600 FCCunits/mg total protein, at least 700 FCC units/mg total protein, atleast 800 FCC units/mg total protein, at least 900 FCC units/mg totalprotein, at least 1000 FCC units/mg total protein, at least 1100 FCCunits/mg total protein, at least 1200 FCC units/mg total protein, atleast 1300 FCC units/mg total protein, at least 1400 FCC units/mg totalprotein, at least 1500 FCC units/mg total protein, at least 1600 FCCunits/mg total protein, at least 1700 FCC units/mg total protein, atleast 1800 FCC units/mg total protein, at least 1900 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 3000 FCCunits/mg total protein, at least 4000 FCC units/mg total protein, atleast 5000 FCC units/mg total protein, at least 6000 FCC units/mg totalprotein, at least 7000 FCC units/mg total protein, at least 8000 FCCunits/mg total protein, at least 9000 FCC units/mg total protein, atleast 10000 FCC units/mg total protein, at least 11000 FCC units/mgtotal protein, at least 12000 FCC units/mg total protein, at least 13000FCC units/mg total protein, at least 14000 FCC units/mg total protein,at least 15000 FCC units/mg total protein, at least 16000 FCC units/mgtotal protein, at least 17000 FCC units/mg total protein, at least 18000FCC units/mg total protein, or at least 19000 FCC units/mg total proteinand less than 20000 FCC units/mg total protein. As used herein, an FCCunit (also referred to herein as a pepsin unit) is defined as thatquantity of enzyme that produces the equivalent of 1 μmol of tyrosineper min under the conditions of incubating the enzyme with 2% hemoglobinsubstrate at pH 1.6 for 10 minutes at 25° C. (see Food Chemical Codex,11th ed. (Pharmacopeial Convention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, the stable pepsin composition comprises arecombinant pepsin having an amino acid sequence of SEQ ID NO. 10 or asequence with at least 90% identity thereto.

In some embodiments, the method further comprising formulating therecombinant pepsin or recombinant pepsinogen with at least oneingredient to create, respectively, a formulated recombinant pepsincomposition or recombinant pepsinogen composition in liquid, powder,pill, tablet, or capsule form. In some embodiments, the liquid is asyrup or a gel

Yet another aspect of the present disclosure is composition comprising arecombinant pepsin polypeptide. The composition is (a) free fromanimal-derived proteins, (b) the pepsin polypeptide is substantially inan intact and stable proteolytically inactive form, (c) the compositionhas a pH greater than about 5.4, and (d) the composition has a specificactivity of between about 100 FCC units/mg total protein and about19,000 FCC units/mg total protein.

In some embodiments, the specific activity at pH of 2 is at least 500FCC units/mg total protein, at least 1000 FCC units/mg total protein, atleast 2000 FCC units/mg total protein, at least 5000 FCC units/mg totalprotein, at least 10000 FCC units/mg total protein, or at least 15000FCC units/mg total protein, and less than 20000 FCC units/mg totalprotein. In some embodiments, the specific activity at pH of 2 is atleast 500 FCC units/mg total protein, at least 1000 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 5000 FCCunits/mg total protein, at least 10000 FCC units/mg total protein, or atleast 15000 FCC units/mg total protein, and less than 20000 FCC units/mgtotal protein. As examples, the specific activity at pH of 2 is at least100 FCC units/mg total protein, at least 200 FCC units/mg total protein,at least 300 FCC units/mg total protein, at least 400 FCC units/mg totalprotein, at least 500 FCC units/mg total protein, at least 600 FCCunits/mg total protein, at least 700 FCC units/mg total protein, atleast 800 FCC units/mg total protein, at least 900 FCC units/mg totalprotein, at least 1000 FCC units/mg total protein, at least 1100 FCCunits/mg total protein, at least 1200 FCC units/mg total protein, atleast 1300 FCC units/mg total protein, at least 1400 FCC units/mg totalprotein, at least 1500 FCC units/mg total protein, at least 1600 FCCunits/mg total protein, at least 1700 FCC units/mg total protein, atleast 1800 FCC units/mg total protein, at least 1900 FCC units/mg totalprotein, at least 2000 FCC units/mg total protein, at least 3000 FCCunits/mg total protein, at least 4000 FCC units/mg total protein, atleast 5000 FCC units/mg total protein, at least 6000 FCC units/mg totalprotein, at least 7000 FCC units/mg total protein, at least 8000 FCCunits/mg total protein, at least 9000 FCC units/mg total protein, atleast 10000 FCC units/mg total protein, at least 11000 FCC units/mgtotal protein, at least 12000 FCC units/mg total protein, at least 13000FCC units/mg total protein, at least 14000 FCC units/mg total protein,at least 15000 FCC units/mg total protein, at least 16000 FCC units/mgtotal protein, at least 17000 FCC units/mg total protein, at least 18000FCC units/mg total protein, or at least 19000 FCC units/mg total proteinand less than 20000 FCC units/mg total protein. As used herein, an FCCunit (also referred to herein as a pepsin unit) is defined as thatquantity of enzyme that produces the equivalent of 1 μmol of tyrosineper min under the conditions of incubating the enzyme with 2% hemoglobinsubstrate at pH 1.6 for 10 minutes at 25° C. (see Food Chemical Codex,11th ed. (Pharmacopeial Convention. 2018) at 1386-87 “Pepsin Activity”).

In some embodiments, the composition has a pH between about 5.4 to about6.0.

In some embodiments, the composition comprises a protein content of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% w/w.

In some embodiments, the composition is in powdered form.

In some embodiments, the composition is in liquid form.

In some embodiments, the recombinant pepsin polypeptide is at least 10mg per liter, at least 100 mg per liter, at least 500 mg per liter, atleast 1 g per liter, at least 2 g per liter, at least 5 g per liter, atleast 7 g per liter, at least 10 g per liter, at least 15 g per liter,or at least 20 g per liter. As examples, the recombinant pepsinpolypeptide is at least 10 mg per liter, at least 20 mg per liter, atleast 30 mg per liter, at least 40 mg per liter, at least 50 mg perliter, at least 60 mg per liter, at least 70 mg per liter, at least 80mg per liter, at least 90 mg per liter, at least 100 mg per liter, atleast 200 mg per liter, at least 300 mg per liter, at least 400 mg perliter, at least 500 mg per liter, at least 600 mg per liter, at least700 mg per liter, at least 800 mg per liter, at least 900 mg per liter,at least 1000 mg per liter, at least 1100 mg per liter, at least 1200 mgper liter, at least 1300 mg per liter, at least 1400 mg per liter, atleast 1500 mg per liter, at least 1600 mg per liter, at least 1700 mgper liter, at least 1800 mg per liter, or at least 1900 mg per liter.

In some embodiments, the recombinant pepsin polypeptide comprises anamino acid sequence of a pig, a sheep, a Central European red deer, agoat, a cow, a human, a yak, or a zebu pepsin.

In some embodiments, the recombinant pepsin polypeptide comprises SEQ IDNO: 10, or an amino acid sequence with at least 90% identity thereto.

In some embodiments, the recombinant pepsin polypeptide is produced in ayeast, a filamentous fungi, a saccharomyces species, a bacteria, aPichia species, a Trichoderma species, or an Aspergillus species. Insome embodiments, the recombinant pepsin polypeptide is produced in aPichia sp.

In some embodiments, the liquid form is a syrup or a gel.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w of the protein in thecomposition is recombinant pepsin.

Additional Embodiments

Embodiment 1: A composition comprising a recombinant pepsinogenpolypeptide, wherein the pepsinogen is substantially in a stableproteolytically inactive form.

Embodiment 2: The composition of embodiment 1, wherein the pepsinogenpolypeptide is present in at least 5 g per liter in the composition.

Embodiment 3: The composition of embodiment 1 or embodiment 2, whereinthe pepsinogen polypeptide when converted to a proteolytically activeform has a higher specific activity as compared to native bovine pepsinisolated from bovine stomach or native porcine pepsin isolated fromporcine stomach in the same quantity.

Embodiment 4: The composition of embodiment 3, wherein the specificactivity of the proteolytically active form is at least 2 times, 2.5times or 3 times greater than the native bovine pepsin or native porcinepepsin.

Embodiment 5: The composition of embodiment 1 or embodiment 2, whereinthe pepsinogen polypeptide is converted to a proteolytically active formand the proteolytically active form has a specific activity of at least50000 or 60000 or 70000 FCC units/mg protein.

Embodiment 6: The composition according to any of embodiments 1 to 5,wherein the recombinant pepsinogen polypeptide is produced in a yeast orfilamentous fungi, a saccharomyces species, a bacteria, a Pichiaspecies, a Trichoderma species or an Aspergillus species.

Embodiment 7: The composition of embodiment 6, wherein the recombinantpepsinogen polypeptide is produced in a Pichia species.

Embodiment 8: The composition of embodiment 1, wherein the pepsinogenexhibits stability in the inactive form for at least 6 months.

Embodiment 9: The composition according to any of embodiments 1 to 8,wherein the composition is in powder form.

Embodiment 10: The composition according to any of embodiments 1 to 9,wherein the recombinant pepsinogen polypeptide is a porcine, bovine,ovine, equine or human pepsinogen.

Embodiment 11: The composition according to any of embodiments 1 to 9,wherein the recombinant pepsinogen polypeptide comprises an amino acidsequence of any one of SEQ ID NOs: 1-9, or an amino acid sequence havingat least 80% homology with any one of SEQ ID NOs: 1-9.

Embodiment 12: A method of obtaining high quantities of recombinantpepsinogen in an inactive form comprising: providing a host cellcomprising a nucleic acid encoding a pepsinogen polypeptide, wherein thenucleic acid further includes a segment directing secretion of thepepsinogen polypeptide from the cell; growing the host cell in a liquidmedium, such that the pepsinogen polypeptide is expressed and secretedfrom the host cell under conditions whereby the pepsinogen polypeptideis substantially in a proteolytically inactive form; isolating liquidmedium containing the secreted pepsinogen polypeptide.

Embodiment 13: The method of embodiment 12, wherein the host cellfurther comprises an inducible promoter driving the expression of thenucleic acid encoding the pepsinogen polypeptide.

Embodiment 14: The method of embodiment 2, wherein the method furthercomprises a step of inducing the expression of the pepsinogen subsequentto or at least partially concurrent with the growing step.

Embodiment 15: The method of embodiment 13 or embodiment 14, wherein thepromoter driving the pepsinogen expression is induced by methanol.

Embodiment 16: The method according to any of embodiments 12-15, furthercomprising treating the isolated liquid media to adjust the pH to about2-4 followed by an adjustment of the pH to 5.5-7.0, e.g., pH 6.

Embodiment 17: The method of embodiment 16, wherein the isolated liquidmedia is filtered at one or more points selected from the groupconsisting of (i) prior to adjusting the pH to 3.5, (ii) after adjustingthe pH to 6.0, (iii) after a desalting step; (iv) prior tolyophilization and (v) a combination of any of (i)-(iv).

Embodiment 18: The method of embodiment 12, wherein the pepsinpolypeptide is present in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 g/liter in theliquid media.

Embodiment 19: The method according to any of embodiments 12-18, whereinthe method further comprises the activation to an enzymatically activeor mature form of the enzyme.

Embodiment 20: The method according to embodiment 19, wherein thepepsinogen polypeptide when converted to an enzymatically active formhas a higher specific activity as compared to native bovine pepsinisolated from bovine stomach or native porcine pepsin isolated fromporcine stomach in the same quantity.

Embodiment 21: The method of embodiment 19, wherein the specificactivity of the enzymatically active form is at least 2 times, 2.5 timesor 3 times greater than the native bovine pepsin or native porcinepepsin.

Embodiment 22: The method according to any of embodiments 12-20, whereinthe pepsinogen polypeptide is converted to an enzymatically active formand the enzymatically active form has a specific activity of at least50000 or 60000 or 70000 FCC units/mg protein.

Embodiment 23: The method according to any of embodiments 12-21, whereinthe host cell is a yeast or filamentous fungi, a saccharomyces species,a bacteria, a Pichia species, a Trichoderma species or an aspergillusspecies.

Embodiment 24: The method of embodiment 22, wherein the host cell is aPichia species.

Embodiment 25: The method according to any of embodiments 12-23, whereinthe pepsinogen polypeptide exhibits stability in the inactive orimmature form for at least 6 months.

Embodiment 26: The method according to any of embodiments 12-24, whereinthe composition is in powder form.

Embodiment 27: The method according to any of embodiments 12-25, whereinthe recombinant pepsinogen polypeptide is a porcine, bovine, ovine,equine or human pepsinogen.

Embodiment 28: The method according to any of embodiments 12-25, whereinthe recombinant pepsinogen polypeptide comprises an amino acid sequenceof any one of SEQ ID NOs: 1-9, or an amino acid sequence having at least80% homology with any one of SEQ ID NOs: 1-9.

Embodiment 29: A formulated composition comprising a recombinantpepsinogen and at least one formulated ingredient, wherein theformulated composition is in a form selected from the group consistingof a liquid (e.g., syrup and gel), powder, a pill, a tablet and acapsule, a microencapsulate, a liposome suspended in syrup and whereinthe pepsinogen is substantially enzymatically inactive.

Embodiment 30: The composition of embodiment 28, wherein the formulatedcomposition is substantially devoid of pepsin.

Embodiment 31: The composition of embodiment 29, wherein the amount ofpepsin is less than 10%, 5%, 1%, 0.5%, 0.1% or 0.05% (weightpepsin/weight pepsinogen).

Embodiment 32: The composition of embodiment 28, wherein the pepsinogenis capable of activation when exposed to a pH less than about 6.

Embodiment 33: The formulated composition of embodiment 31, wherein thepH of the composition is greater than about 6.

Embodiment 34: The formulated composition of embodiment 31, wherein thepepsinogen is capable of activation when exposed to an animal gutenvironment.

Embodiment 35: The formulated composition of embodiment 28, wherein therecombinant pepsinogen is a bovine, porcine, ovine or human enzyme.

Embodiment 36: A method of preparing a formulated pepsinogen compositioncomprising: providing a recombinant pepsinogen, wherein the pepsinogenis substantially enzymatically inactive; formulating the recombinantpepsinogen with at least one ingredient to create a formulatedpepsinogen composition in liquid (e.g., syrup and gel), powder, pill,tablet or capsule form: wherein the formulated pepsinogen composition iscapable of activation when exposed to a pH of less than about 6.

Embodiment 37: The method of embodiment 35, wherein the recombinantpepsinogen is a bovine, porcine, ovine or human enzyme.

Embodiment 38: The method of embodiment 35, wherein the recombinantpepsinogen is produced in a heterologous host cell selected from thegroup consisting of bacteria, yeast, and filamentous fungi.

Embodiment 39: The method of embodiment 35, wherein the host cell is aPichia species, a saccharomyces species, a bacteria, an Aspergillus or aTrichoderma species.

Embodiment 40: The method of embodiment 36-39, wherein the recombinantpepsinogen is secreted from the host cell.

Embodiment 41: The method of embodiment 40, wherein the recombinantpepsinogen secreted is in a substantially enzymatically inactive form.

Embodiment 42: A composition comprising recombinant stable pepsin,wherein the stable pepsin is substantially enzymatically inactive.

Embodiment 43: The composition of embodiment 42, wherein recombinantstable pepsin is capable of activation when exposed to a pH less thanabout 6.

Embodiment 44: The composition of embodiment 42, wherein the pH of thecomposition is greater than about 6.

Embodiment 45: The composition of embodiment 42, wherein the recombinantstable pepsin is capable of activation when exposed to an animal gutenvironment.

Embodiment 46: The composition of embodiment 42, wherein the recombinantstable pepsin is a bovine, porcine, ovine or human enzyme.

Embodiment 47: The composition according to any of embodiments 42-46,wherein the specific activity of the recombinant stable pepsin whenactivated to a proteolytically active form is at least 2 times, 2.5times or 3 times greater than the native bovine pepsin or native porcinepepsin.

Embodiment 48: The composition according to any of embodiments 42-46,wherein the recombinant stable pepsin when converted to aproteolytically active form has a specific activity of at least 50000 or60000 or 70000 FCC units/mg protein.

Embodiment 49: The composition according to any of embodiments 42-48,wherein the recombinant stable pepsin is first produced as a pepsinogenpolypeptide in a yeast or filamentous fungi, a saccharomyces species, abacteria, a Pichia species, a Trichoderma species or an Aspergillusspecies and then converted to pepsin.

Embodiment 50: A method of treating a disease or condition of thegastrointestinal tract comprising: providing a recombinant pepsinogen ina formulated composition, wherein the pepsinogen is substantiallyenzymatically inactive in the formulated composition; administering theformulated composition for oral administration; wherein upon contact ofthe formulated composition with an animal gut environment, thepepsinogen is converted to an enzymatically active form; and wherein theenzymatically active form is effective to treat the disease or conditionof the gastrointestinal tract.

Embodiment 51: The composition of any of embodiments 1-11, wherein thecomposition comprises at least one production specification set forth inTable 2.

Embodiment 52: The composition of any of embodiments 1-11 or embodiment51, wherein the composition comprises at least one quality specificationset forth in Table 3.

Definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting.

As used herein, unless otherwise indicated, the terms “a”, “an” and“the” are intended to include the plural forms as well as the singleforms, unless the context clearly indicates otherwise.

The terms “comprise”, “comprising”, “contain,” “containing,”“including”, “includes”, “having”, “has”, “with”, or variants thereof asused in either the present disclosure and/or in the claims, are intendedto be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean 10% greater than or less than the statedvalue. In another example, “about” can mean within 1 or more than 1standard deviation, per the practice in the given value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” should be assumed to mean anacceptable error range for the particular value.

The term “substantially” is meant to be a significant extent, for themost part; or essentially. In other words, the term substantially maymean nearly exact to the desired attribute or slightly different fromthe exact attribute. Substantially may be indistinguishable from thedesired attribute. Substantially may be distinguishable from the desiredattribute but the difference is unimportant or negligible.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of various embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Expression of Pepsinogen

The coding sequence of porcine pepsinogen (SEQ ID NO: 2) was fused tothe saccharomyces alpha factor pre-pro secretion signal under thecontrol of a methanol-induced promoter. The fusion was constructed suchthat the pro form, pepsinogen, lacking the native secretion signal wasproduced upon expression and secretion of the pepsinogen.

The P. pastoris strain BG08 (BioGrammatics Inc., Carlsbad; Calif., USA)is a single colony isolate from the Phillips Petroleum strain NRRLY-11430 obtained from the Agriculture Research Service culturecollection (Sturmberger, et al. 2016). P. pastoris BG10 (BioGrammaticsInc, Carlsbad, Calif., USA) was derived from BG08 using Hoechst dyeselection to remove cytoplasmic killer plasmids (Sturmberger, et al.2016). This BG10 strain was then further modified to have a deletion inthe Alcohol Oxidase 1 gene (AOX1). This deletion generates amethanol-utilization slow (mutS) phenotype that reduces the strain'sability to consume methanol, e.g., as a carbon source. This base strainwas called DFB-001 and used for the transformation of the pepsinogenconstruct.

The pepsinogen construct, along with a construct for the expression ofthe P. pastoris transcription factor HAC1 under the control of a strongmethanol inducible promoter, was transformed into Pichia pastoris andisolates were selected that expressed and secreted pepsinogen. Atransformant was selected as a high-producer for use in subsequentsteps. Propagation of the strain confirmed that all changes introducedinto the strain were stably integrated in the genome and confirmed to bepresent after >45 generations of growth on non-selective growth media.

Sequencing confirmed that this strain does not contain any antibioticmarkers or prokaryotic vector origin of replication sequences.

The resulting strain was grown in fermentation conditions inhigh-density growth conditions at about pH 5. After about 36 hours ofgrowth under fermentation conditions, the pH was raised to about pH 6,and expression of pepsinogen was induced by the addition of methanol tothe culture. The pepsinogen was isolated from the growth media of theculture.

In some cases, the liquid media was centrifuged to remove P. pastoriscells. This was followed by filtration of the supernatant using a 0.2 umhollow fiber membrane filtration to remove host protein and cell debris.

Example 2: Conversion of Pepsinogen to Pepsin

The pepsinogen solution was concentrated to a 5× to 10× concentrateusing 10 kDa hollow fiber membrane filtration. An acid solution of 85%phosphoric acid was added to the resulting liquid composition fromExample 1 to lower the pH to 3.5, and the mixture was agitated for 2.25hours at room temperature (about 20-25° C.). Following this agitationstep, the pH was raised to 6 by addition of 5 N NaOH. The resultingconcentrate was desalted with 10 DV of distilled water at pH 6 using 10kDa hollow fiber membrane filtration. Then the solution was lyophilizedto produce a pepsin powder.

Example 3: Characterization of Pepsinogen Activation

The pepsinogen composition (from Example 1 or 2) was characterized forits activation post dilution, e.g., conversion to active pepsin, undervarious temperature and pH ranges. In FIG. 1, the pepsinogen compositionat pH 5.95 (B lane) was shifted to pH 3.5 by the addition of 2.5 N HCland incubated for the times shown at room temperature (about 20°-25°C.), 35° C. or 45° C.; samples were raised to pH 6 using 2 NaOH. Sampleswere analyzed using polyacrylamide gel electrophoresis (PAGE) andconversion of pepsinogen to pepsin was observed as a change in theapparent molecular of the protein.

FIG. 2, shows that the pepsinogen composition at pH 5.95 (B lane) wasshifted to pH 1.5, 2.5, or 3.5 by the addition of 2.5 N HCl andincubated at 37° C. for the times shown followed by pH raising to pH 6.0with 2 NaOH. Samples were analyzed using PAGE and conversion ofpepsinogen to pepsin observed as a change in the apparent molecularweight of the protein. FIG. 3 shows images of protein gels of apepsinogen composition for the times shown (in hours) at pH 1.5, 2.5,3.5, and at room temperature; samples were raised to pH 6 using 2 NaOH.Samples were analyzed using PAGE and conversion of pepsinogen to pepsinwas observed as a change in the apparent molecular of the protein.

FIG. 4, shows the pepsinogen composition activated at temperatures of45° C. and 55° C. for the times shown, and raised to pH 6 using 2 NaOH.Samples were analyzed using PAGE and conversion of pepsinogen to pepsinwas observed as a change in the apparent molecular of the protein.

Example 4: Characterization of Recombinant Pepsin

Lyophilized pepsin powder (Example 2) was subjected to a proteindetermination using combustion analysis (N X 6.25) and activity wasmeasured in an FCC9 enzyme assay (USP, Pepsin activity. In: NinthEdition of the Food Chemicals Codex (FCC 9). United States PharmacopeiaConvention, Rockville, Md., 2015e, pp. 1410-1411; see also Food ChemicalCodex, 11th ed. (Pharmacopeial Convention. 2018) at 1386-87 “PepsinActivity”). As compared to a commercially-available porcine pepsinproduct, the recombinant pepsin composition had over 3-times thespecific activity.

Table 1 shows the results of the analysis for protein, fat, moisture,ash, carbohydrates and FCC units for the commercially-available nativeporcine pepsin and for a composition comprising recombinant pepsin. FCCunits/mg is defined as follows: One pepsin unit is defined as thatquantity of enzyme that produces the equivalent of 1 μmol of tyrosineper min under the conditions of incubating the enzyme with a 2%hemoglobin substrate at pH 1.6 for 10 minutes at 25° C. performed as setforth in Food Chemical Codex, 11th ed. (Pharmacopeial Convention. 2018)at 1386-87 “Pepsin Activity” (the same assay is also provided in NinthEdition of the Food Chemicals Codex (FCC 9). United States PharmacopeiaConvention, Rockville, Md., 2015e, pp. 1410-1411).

TABLE 1 FCC unit comparison between a commercially-available pepsin anda recombinant pepsin composition. Commercial Recombinant Pepsin PorcinePepsin Composition Protein % w/w (N X 6.25) 50.25 31.3 Fat % w/w 0.5350.285 Moisture % w/w 4.4 7.3 Ash % w/w 1.7 4.95 Carbs (by difference) %w/w 43.11 56.15 Activity FCC units/mg protein 27,343 70,863

Surprisingly, while the percentage of protein in the recombinant pepsincomposition is significantly lower than the commercially-availablenative porcine pepsin, the FCC units of the recombinant pepsinogencomposition is significantly (about 3×) higher. Without wishing to bebound by theory, the methods for manufacturing the pepsin compositionsof the present disclosure provide a highly active product.

Example 5: Comparison of Pepsin Activity Profiles

A recombinant pepsin composition was tested for activity against a rangeof pH and compared against the activity of native porcine pepsin (seeExample 4) using the FCC (9th Edition) Pepsin assay (PharmacopeialConvention. 2014). The optimum activity was at pH 2 for both nativeporcine pepsin and the recombinant pepsin polypeptide of the presentdisclosure. Both pepsin enzymes tested had a similar activity profile(FIG. 5A and FIG. 5B). The pepsin activity in FIG. 5A is presented asFCC units/units pepsin, wherein each Pepsin unit is defined as theamount of pepsin present in the sample, derived from its peak areadetermined through HPLC. The pepsin activity in FIG. 5B is presented asFCC units/mg powder.

In some cases, pepsin assays were performed with the following changes(“alternate pepsin assay”) to the assay described in Example 4: Theactivity assay was performed at 37° C. in a 96-well plate format andtyrosine was measured directly. These changes result in an output numberthat when multiplied by two (2) is equivalent to the FCC units ofactivity performed as in Example 4. The numbers reported for allalternate pepsin assays herein apply this conversion factor.

Example 6: Comparison of Immunoreactivity and Molecular Weights

A recombinant pepsin composition and native porcine pepsin (see Example4) were compared for immunoreactivity and molecular weight using westernblotting technique (Tobin). Briefly, three separately-generated lots ofthe recombinant pepsin composition (PEP19232, PEP19241, PEP19252) andnative porcine pepsin were run on SDS-PAGE and then transferred tonitrocellulose membrane. Western Blot was performed on the samples usingprimary pepsin antibody from rabbit (Abcam (ab182945)) at a 1:5000dilution (Jensen 2012). The secondary antibody used was Goat anti-rabbitIgG conjugated to alkaline phosphatase (1:5000 dilution). FIG. 6 showsthe western blot comparing the proteins.

Example 7: Pepsin Specifications

Based upon the characterization of the recombinant pepsin compositionsand the properties of commercially-available native porcine pepsin,product specifications (Table 2) and quality control specifications(Table 3) were constructed.

TABLE 2 Product specifications for a recombinant pepsin compositionPhysical properties Specification Source Yeast fermentation-derivedAppearance White to off-white amorphous powder Solubility Mostly solublein water with slight opalescence. Practically insoluble in alcohol,chloroform and ether. Specification Method Enzyme Activity Activity inUnits/mg 1:30000 FCC Assay¹ powder FCC Units Chemical Properties (inpowder as is) Moisture Maximum 10.0% AOAC 925.09² Ash Maximum 5.0% AOAC942.05³ Hg <1 ppm ICP-AES⁴ Pb <1 ppm ICP-AES⁴ As <1 ppm ICP-AES⁴ Cd <1ppm ICP-AES⁴ Microbial Properties (in powder as is) Standard Plate Count<10000 CFU/g AOAC 990.12⁵ Yeast & Mold <100 CFU/g AOAC 997.02⁶Salmonella Not Detected/25 g AOAC 2003.09⁷ E. coli <10 CFU/g AOAC991.14⁸ Total coliform <30 CFU/g AOAC 991.14⁸ ¹Food Chemical Codex, 9thed. (Pharmacopeial Convention. 2014) ²Association of Official AnalyticalChemists (1995). In Official Methods of Analysis. ³J AOAC Int. 2012September-October; 95(5): 1392-7. ⁴J. AOAC vol. 90 (2007) 844-856. ⁵AOACInternational (2005). Aerobic plate count in foods, dry rehydratablefilm, method 990.12. AOAC International, 17th ed. Gaithersburg, MD.⁶17.2.09 AOAC Official Method 997.02. Yeast and Mold Counts in Foods DryRehydratable Film Method (Petrifilm ™ Method) First Action 1997 FinalAction 2000 ⁷AOAC International. 2005. Salmonella in selected foods, BAXautomated system, method 2003.09. In Official methods of analysis ofAOAC International, 17th ed. AOAC International, Gaithersburg, MD. ⁸AOACInternational. 2005, E. coli count in foods, dry rehydratable film,method 991.14. In Official methods of analysis of AOAC international,17th ed. AOAC International, Gaithersburg, MD.

TABLE 3 Quality specifications for recombinant pepsin compositionsParameter Specification * PEP19232 PEP19241 PEP19252 Activity (FCC1:30000 1:31440 1:31000 1:32200 Units/mg powder) Moisture <10% 9.4 9.19.6 Ash %  <5% 3.54 3.79 3.61 Hg <1 mg/kg <0.01 <0.01 <0.01 Pb <1 mg/kg<0.01 <0.01 <0.01 As <1 mg/kg <0.01 <0.01 <0.01 Cd <1 mg/kg <0.01 <0.01<0.01 Aerobic plate count <10000 CFU/g <10 <10 <10 Yeast & Mold <100CFU/g <10 <10 <10 Salmonella Not Detected/25 g Not detected Not detectedNot detected E. coli <10 CFU/g <10 <10 <10 Total coliforms ≤30 CFU/g Notdetected Not detected Not detected Absence of source Not detected/ Notdetected Not detected Not detected organism from mg sample productAbsence of Not detected ^(#)/ Not detected Not detected Not detectedencoding DNA mg sample from product

Example 8: Absence of DNA in Recombinant Pepsin Compositions

In this example, experiments were performed to confirm the absence oftransformable DNA in the recombinant pepsin preparation made andisolated from the Pichia strain.

Materials: 2× Taq MasterMix from NEB; Primers:5′GAAGCTGAAGCTCTAGTAAAGGTGCCTCTAG (forward; SEQ ID NO: 12); 5′TGCAACAGGTGCTAGACCCACCTTGTTGTTAG (reverse; SEQ ID NO: 13). The primershave an annealing temp of 58° C. when using 2× Taq MasterMix); controlDNA is the pepsinogen transformation cassette (Example 1).

Methods: A pepsin composition in powdered form (see above Examples) wasdiluted to 100 mg powder/mL in 25 mM sodium hydroxide and then 250 μLwas transferred to two new tubes with 2504, of 25 mM sodium hydroxide(this created two new 500 μL tubes of 50 mg/mL pepsin). To one of thesetubes, a positive control pepsinogen plasmid DNA was added to get afinal concentration of 1 ng/μL (this served as a positive control forthe assay). This tube was then used in sequential dilutions to generatea series of controls with the lowest concentration at 1 fg of controlDNA. PCR reactions using the forward and reverse primers (of SEQ ID NOs:12 and 13) were then performed on the powdered pepsin composition(without control DNA) and the series containing powdered pepsincompositions (with control DNA). PCR products were run on a 1% agarosegel. PCR product for pepsin (as shown in the control lanes) produced a1122 bp band. This band was absent in each of the three powdered pepsincomposition lots tested (see FIG. 7A to FIG. 7C). The detection limit ofthe assay was about 1 fg of pepsin DNA.

Example 9: Demonstration of Absence of Host Cells in Recombinant PepsinCompositions

Materials: Minimal methanol (MM) agar plates; Potato Glucose Agar (PGA)plates Procedure: Powdered recombinant pepsin compositions were platedon PGA plates. If samples yielded colonies, partial samples of thecolony were streaked onto PGA plates and MM plates and incubated asfollows: PGA plates for 48 hours at 30° C.; MM plates for 120 hours at30° C. If colonies grew on MM plates within 120 hours at 30° C., singlecolonies were picked and colony PCR with cassette specific primers wasrun. (see PCR method, Example 8). If colony PCR confirmed the presenceof the pepsinogen expression cassette, it could be concluded thatrecombinant pichia cells are present in the pepsin composition.

This procedure was applied to three lots of powdered pepsin compositionproduced from the recombinant strain (Example 1). No recombinant pichiacells were detected in any of the lots (see Table 3 “source organism”set forth in Example 7).

Example 10: Comparison of the Purity of a Recombinant Pepsin Compositionand a Commercially-Available Native Porcine Pepsin

A recombinant pepsin composition was compared to native porcine Pepsin Aby liquid chromatography tandem mass spectrometry (LC-MS/MS). Theprotein samples were first digested into peptides using endoproteinaseGluC or chymotrypsin, in parallel, to get improved cleavage of Pepsin A.The peptides produced were analyzed through LC-MS/MS. The resultingspectra were matched to peptide sequences using the software tool,X!tandem (see the World Wide Web at:proteomics.ucdavis.edu/protein-identification/). A summary of theproteins present by category is shown in Table 4. The results from thechymotrypsin digest are presented in Table 5.

TABLE 4 Protein abundance and impurities (non-target proteins) Valuesshown are for Exponentially Modified Protein Abundance Index (emPAI)Porcine Recombinant Accession Number Pepsin Pepsin Porcine Pepsin(PEPA_PIG; 826.8 1280.1 SEQ ID NO: 10) Pig protein (I3LL32_PIG) 822.80.0 Pig Protein (F1S636_PIG) 491.4 0.0 Miscellaneous proteins 18.9 12.7

The LC-MS/MS data for the recombinant pepsin exactly matched the matureform of native Sus scrofa (Porcine) Pepsin A (PEPA_PIG; SEQ ID NO 10).

Based on the LC-MS/MS results, recombinant pepsin (“rPepsin”) was foundto contain an abundance of the PEPA_PIG porcine pepsin sequence (Table 4and FIG. 8).

The native porcine pepsin contained PEPA_PIG protein and othercontaminating porcine proteins in high abundance whereas the recombinantpepsin compositions did not have as many protein impurities (Table 5 andFIG. 8).

TABLE 5 LC-MS/MS Protein Abundance data Mol. Wt Porcine RecombinantIdentified Proteins Accession Number (kDa) Pepsin pepsin Cluster ofPepsin A OS = Sus scrofa PEPA_PIG [2] 41 826.8 1280.1 OX = 9823 GN = PGAPE = 1 SV = 3 (PEPA_PIG; SEQ ID NO: 10) Cluster of Chymotrypsinogen A OS= Bos CTRA_BOVIN [3] 26 275.3 1273.6 taurus PE = 1 SV = 1 (CTRA_BOVIN)Uncharacterized protein OS = Sus scrofa I3LL32_PIG 52 822.8 0.0 OX =9823 GN = CHIA PE = 3 SV = 2 Uncharacterized protein OS = Sus scrofaF1S636_PIG 44 491.4 0.0 OX = 9823 GN = LOC100620249 PE = 3 SV = 2Lectin-like protein with similarity to C4QYW7_KOMPG 51 0.0 6.9 Flo1p,thought to be expressed and involved in flocculation OS = Komagataellaphaffii (strain GS115/ ATCC 20864) OX = 644223 GN = PAS_chr1-4_0584 PE =4 SV = 1 Cluster of Uncharacterized protein F1SCD0_PIG [2] 47 6.7 0.0 OS= Sus scrofa OX = 9823 GN = LOC100153899 PE = 1 SV = 1 (F1SCD0_PIG)Cluster of Uncharacterized protein A0A287AVA9_PIG 47 3.4 0.0 OS = Susscrofa OX = 9823 [3] GN = LOC100153899 PE = 1 SV = 1 (A0A287AVA9_PIG)Cluster of Fibrillin-1 OS = Sus scrofa F1SN67_PIG [2] 312 0.3 0.0 OX =9823 GN = FBN1 PE = 1 SV = 3 (F1SN67_PIG) Uncharacterized protein OS =Sus scrofa A0A287ANG4_PIG 440 0.1 0.0 OX = 9823 GN = MUC5AC PE = 1 SV =1 Gastricsin OS = Sus scrofa OX = 9823 A0A286ZP41_PIG 43 3.6 0.0 GN =PGC PE = 3 SV = 1 (+1) Uncharacterized protein OS = Sus scrofaI3LHI7_PIG 28 1.7 0.0 OX = 9823 GN = LOC100621820 PE = 3 SV = 2 ContainsGLEYA adhesin domain C4R9C9_KOMPG 110 0.0 0.3 OS = Komagataella phaffii(strain GS115/ ATCC 20864) OX = 644223 GN = PAS_c034_0002 PE = 4 SV = 1Fibrillin 2 OS = Sus scrofa OX = 9823 F1RKK1_PIG 303 0.1 0.0 GN = FBN2PE = 1 SV = 3 Mucin 6, oligomeric mucus/gel-forming I3LQZ3_PIG 200 0.10.0 OS = Sus scrofa OX = 9823 GN = MUC6 PE = 4 SV = 2 Cluster ofFibrillin 3 OS = Sus scrofa I3LI73_PIG [2] 304 0.0 0.0 OX = 9823 GN =FBN3 PE = 1 SV = 2 (I3LI73_PIG) Uncharacterized protein C4R3B1_KOMPG 410.0 1.7 OS = Komagataella phaffii (strain GS115/ ATCC 20864) OX = 644223GN = PAS_chr3_0016 PE = 4 SV = 1 Uncharacterized protein OS = Sus scrofaI3LJ52_PIG 28 0.5 0.0 OX = 9823 GN = CTRB2 PE = 3 SV = 1Phosphatidylglycerol/phosphatidylinositol C4QZC2_KOMPG 19 0.0 2.7transfer protein OS = Komagataella phaffii (strain GS115/ATCC 20864) OX= 644223 GN = PAS_FragB_0077 PE = 4 SV = 1 Serpin family F member 2 OS =Sus A0A287AJI4_PIG 53 0.2 0.0 scrofa OX = 9823 GN = SERPINF2 PE = 1 (+1)SV = 1 Essential component of the nuclear pore C4R4T7_KOMPG 105 0.1 0.0complex OS = Komagataella phaffii (strain GS115/ATCC 20864) OX = 644223GN = PAS_chr3_0524 PE = 4 SV = 1 Tripeptidyl peptidase 1 OS = Sus scrofaA0A287AM42_PIG 62 0.3 0.0 OX = 9823 GN = TPP1 PE = 1 SV = 1 (+2)Uncharacterized protein OS = Sus scrofa F6Q469_PIG 46 0.8 0.0 OX = 9823GN = SERPINA3-2 PE = 1 SV = 1 Uncharacterized protein C4R3C4_KOMPG 630.0 0.1 OS = Komagataella phaffii (strain GS115/ ATCC 20864) OX = 644223GN = PAS_chr3_0030 PE = 4 SV = 1 Uncharacterized protein OS = Sus scrofaA0A287BM11_PIG 45 0.7 0.0 OX = 9823 GN = LOC396684 PE = 1 SV = 1 (+1)Thioredoxin OS = Komagataella phaffii C4R7E5_KOMPG 11 0.0 0.9 (strainGS115/ATCC 20864) OX = 644223 GN = PAS_chr4-0284 PE = 3 SV = 1Ribonuclease K3 OS = Sus scrofa RNAS6_PIG 17 0.4 0.0 OX = 9823 GN =RNASE6 PE = 1 SV = 2

Without wishing to be bound by theory, the methods for manufacturing thepepsin compositions of the present disclosure provide a highly activeproduct which is free from animal-derived proteins, and with low (or nodetectable) amounts of host cell proteins.

Example 11: Analysis of Stability of a Powdered Recombinant PepsinComposition

The objective of this analysis was to determine the stability of apowdered recombinant composition. Here, a composition was diluted withcommon salt, under room temperature and refrigerated storage conditions.

Material and Methods:

Sample preparation: a powdered recombinant pepsin composition (Lot#PEP19225) was diluted with sodium chloride (Micro powder salt flour, TheGreat American Spice Company) to achieve an activity of 10000 FCCUnits/mg powder. After thorough mixing, the diluted composition wasaliquoted into 25 Kraft barrier pouches (FDA and USDA compliant) andsealed.

One pouch was sent for analysis for baseline data (Time Point TP 0).Twelve pouches were stored at about 4° C. and another twelve were storedat room temperature. Samples were pulled from each of the two storageconditions at monthly intervals and sent for analyses.

Tests conducted:

-   -   1. Activity (FCC Units/mg powder) following the Pepsin Assay        method by Food Chemical Codex, 9th ed. (Pharmacopeial        Convention. 2014).    -   2. Moisture % using method AOAC 925.09, Association of Official        Analytical Chemists (1995). In Official Methods of Analysis.    -   3. Aerobic Plate Count using method AOAC 990.12 (AOAC        International (2005). Aerobic plate count in foods, dry        rehydratable film, method 990.12. AOAC International, 17th ed.        Gaithersburg, Md.)    -   4. Yeast and mold using 17.2.09 AOAC Official Method 997.02.        Yeast and Mold Counts in Foods Dry Rehydratable Film Method        (Petrifilm™ Method) First Action 1997 Final Action 2000

Results for the first seven months of the room temperature (18° C. to20° C.) study are shown below in Table 6 and results for the first fivemonths of the refrigerated (˜4° C.) study are shown below in Table 7.

TABLE 6 Time Point Method Specification 0 1 2 3 4 5 6 7 Activity FCC10000 (FCC/ 10708 10722 9218 9512 10412 10746 11974 10996 (FCC Pepsin mgpowder) Units/mg Assay powder) Aerobic Plate AOAC <10000 CFU/g  20 <1020 20 <10 <10 <10 15 Count 990.12 Yeast AOAC <100 CFU/g <10 <10 <10 <10<10 <10 <10 <10 997.02 mold AOAC <100 CFU/g <10 10 <10 <10 <10 <10 <10<10 997.02 Moisture AOAC <10% 4.5 n/a 5.2 4.75 4.45 4.57 4.6 4.98 925.09

TABLE 7 Time Point Method Specification 0 1 2 3 4 5 6 7 Activity FCC10000 10708 11188 10070 10040 10452 10270 11512 11044 (FCC/mg Pepsin(FCC/mg powder) Assay powder) TPC AOAC <10000 20 40 <10 65 <10 <10 <1020 990.12 CFU/g Yeast AOAC <100 <10 <10 <10 <10 <10 <10 <10 <10 997.02CFU/g mold AOAC <100 <10 <10 <10 <10 <10 <10 <10 <10 997.02 CFU/gmoisture AOAC <10% 4.5 n/a 4.7 4.47 3.98 3.93 4.0 4.53 925.09

These data show that the microbial load on the samples over the firstseven months was well within acceptable limits for both refrigerated androom temperature storage conditions. The recombinant pepsin activity wasstable for at least seven months under refrigerated storage and at roomtemperature.

Without wishing to be bound by theory, the methods for manufacturing thepowdered pepsin compositions of the present disclosure provide a highlystable product.

Example 12: Analysis of Stability of a Liquid Recombinant PepsinComposition

The objective of this analysis was to determine the stability of aliquid recombinant composition. Here, a composition was diluted withphosphate citrate buffer, under refrigerated storage conditions over aperiod of time.

Material and Methods:

A powdered recombinant pepsin composition (GRAS Test Lot #2 (CS462) wasobtained and diluted in 0.01 M phosphate citrate buffer, pH 6.0, toproduce a composition comprising 1% recombinant pepsin. The compositionwas aliquoted into 50 ml conical tube and stored in a 4° C.refrigerator.

Samples were collected every two weeks and pepsin activity was assayed;using an assay based on the Worthington Pepsin Assay: hemoglobinsubstrate, pH 1.6, 37° C., 10 minutes.

Data is shown below in Table 8 and FIG. 9.

TABLE 8 Days Activity at 4° C. (U/mg) 0 9757 14 10135 28 8970 46 8898

These data show that the liquid recombinant pepsin composition hadmaintained activity within the variation of the pepsin activity assay(15%) over the course of 46 days at 4° C. Thus, the recombinant pepsincomposition can be considered liquid-stable at pH 6.

Without wishing to be bound by theory, the methods for manufacturing theliquid pepsin compositions of the present disclosure provide a highlystable product.

While embodiments of the present invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A composition comprising a recombinant pepsinpolypeptide, wherein (a) the composition is free from animal-derivedproteins, (b) the pepsin polypeptide is in a proteolytic ally inactiveform which does not self-digest, (c) the composition has a pH of 5.4 orhigher, and (d) the composition has a specific activity of from 500 FCCunits/mg total protein to less than 30,000 FCC units/mg total protein;wherein the composition is in powdered form.
 2. The composition of claim1, wherein the composition comprising the proteolytically inactivepepsin polypeptide form which does not self-digest is stable for atleast 6 months at room temperature or for at least 6 months at 4° C. 3.The composition of claim 2, wherein the composition has a moisturecontent of less than about 10%.
 4. The composition of claim 1, whereinthe recombinant pepsin polypeptide has a specific activity of from 500FCC units/mg total protein to 20,000 FCC units/mg total protein at pH 2.5. The composition of claim 1, wherein the composition has a pH of 6.0or higher.
 6. The composition of claim 1, wherein the powdered form canbe solubilized in an aqueous medium to create a liquid compositionhaving a concentration of the recombinant pepsin polypeptide that is atleast 20 g per liter.
 7. The composition of claim 6, wherein theproteolytically inactive pepsin polypeptide form which does notself-digest is stable in the liquid composition for at least 30 days ata temperature of 4° C.
 8. The composition of claim 1, wherein therecombinant pepsin polypeptide comprises an amino acid sequence of asheep, pig, cow, human, zebu, yak, Central European red deer, or goatpepsin.
 9. The composition of claim 1, wherein the recombinant pepsinpolypeptide comprises SEQ ID NO: 10, or an amino acid sequence with atleast 90% identity thereto.
 10. The composition of claim 1, wherein therecombinant pepsin polypeptide is produced in a Pichia species, a yeast,a filamentous fungi, a Saccharomyces species, a bacterium, a Trichodermaspecies, or an Aspergillus species.
 11. The composition of claim 1,wherein the composition comprises less than 5% of contaminatingproteins.
 12. The composition of claim 11, wherein the contaminatingprotein is a non-pepsin protein.
 13. The powdered composition of claim1, produced by a method comprising steps of: a. providing amicroorganism expressing a recombinant pepsinogen, wherein the expressedpepsinogen is secreted by the microorganism into a growth media; b.harvesting the growth media and removing the microorganismal cellstherefrom to obtain a liquid starting material; c. lowering the pH ofthe liquid starting material to less than pH 4.0 to obtain an activatedpepsin composition; d. raising the activated pepsin composition to a pHof 5.4 or higher to obtain the composition comprising the recombinantpepsin polypeptide; and e. isolating the recombinant pepsin polypeptidefrom protein and small molecules in the liquid starting material toobtain the powdered composition.
 14. The powdered composition of claim13, wherein the recombinant pepsinogen comprises any one of SEQ ID NO: 1to SEQ ID NO: 9, or an amino acid sequence with at least 90% identitythereto.
 15. A method for producing a high-activity stable pepsinpowdered composition comprising steps of: a. providing a microorganismexpressing a recombinant pepsinogen, wherein the expressed pepsinogen issecreted by the microorganism into its growth media; b. harvesting thegrowth media and removing the microorganismal cells therefrom to obtaina liquid starting material; c. lowering the pH of the liquid startingmaterial to less than pH 4.0 to obtain an activated pepsin composition;d. raising the activated pepsin composition to a pH of 5.4 or higher toobtain a high-activity stable pepsin composition; and e. isolating thestable pepsin polypeptide from protein and small molecules in the liquidstarting material to obtain the high-activity stable pepsin powderedcomposition having a specific activity of less than 10,000 FCC units/mgtotal protein; wherein step (e) is performed after steps (c) and (d).16. The method of claim 15, wherein the high-activity stable pepsincomposition comprises a pepsin polypeptide having an amino acid sequenceof a sheep, pig, cow, human, zebu, yak, Central European red deer, orgoat pepsin.
 17. The method of claim 15, wherein the microorganism is aPichia species, a yeast, a filamentous fungus, a Saccharomyces species,a bacterium, a Trichoderma species, or an Aspergillus species.
 18. Themethod of claim 15, wherein the high-activity stable pepsin powderedcomposition comprises less than 5% of contaminating proteins.
 19. Themethod of claim 18, wherein the contaminating protein is a non-pepsinprotein.
 20. The method of claim 15, wherein the recombinant pepsinogencomprises any one of SEQ ID NO: 1 to SEQ ID NO: 9, or an amino acidsequence with at least 90% identity thereto.
 21. The method of claim 15,wherein the high-activity stable pepsin powdered composition comprisesat least 500 FCC units/mg total protein.
 22. A method for producing aconsumable composition, the method comprising: providing thehigh-activity stable pepsin powdered composition of claim 1; andcombining the high-activity stable pepsin powdered composition with afood or beverage ingredient, thereby producing a consumable composition.23. A food or beverage product comprising the high-activity stablepepsin powdered composition of claim 1 and a food or beverageingredient.